Odilia Coi

Guillaume Patrigeon

Sophiane Senni

Lionel Torres

Pascal Benoit

A novel SRAM -STT-MRAM hybrid cache implementation improving cache performance

Memories are currently a real bottleneck to design high speed and energy-efficient systems-on-chip. A significant increase of the performance gap between processors and memories is observed. On the other hand, an important proportion of total power is spent on memory systems due to the increasing trend of embedding volatile memory into systems-on-chip. For these reasons, STT-MRAM (Spin-Transfer Torque Magnetic Random Access Memory) is seen as a promising alternative solution to traditional SRAM (Static Random Access Memory) thanks to its negligible leakage current, high density, and non-volatility. Nevertheless, the strategy of the same footprint replacement is constrained by the high write energy/latency of STT-MRAM. This paper performs a fine-grained evaluation of the cache organization to propose a hybrid cache memory architecture including both SRAM and STT-MRAM technologies.

I. INTRODUCTION AND RELATED WORKS

Several studies, for instance in [START_REF] Xu | Design of Last-Level On-Chip Cache Using Spin-Torque Transfer RAM (STT RAM)[END_REF], [START_REF] Wu | Hybrid cache architecture with disparate memory technologies[END_REF], [START_REF] Wu | Power and performance of read-write aware hybrid caches with non-volatile memories[END_REF], explored the combination of STT-MRAM and SRAM to propose fast and low-power cache memories. Novel management techniques have been proposed to mitigate the high write energy/latency of STT-MRAM based cache such as in [START_REF] Zhou | Energy reduction for STT-RAM using early write termination[END_REF], [START_REF] Kwon | Aware (asymmetric write architecture with redundant blocks): A high write speed STT-MRAM cache architecture[END_REF], [START_REF] Wang | OAP: An obstruction-aware cache management policy for STT-RAM last-level caches[END_REF]. Other works explored the performance improvement of STT-MRAM by reducing the retention time [START_REF] Cheng | Architecture and data migration methodology for L1 cache design with hybrid SRAM and volatile STT-RAM configuration[END_REF], [START_REF] Jog | Cache revive: Architecting volatile STT-RAM caches for enhanced performance in CMPs[END_REF].

This paper evaluates a novel hybrid cache architecture by building the tag arrays and the data arrays with two different memory technologies (SRAM and STT-MRAM). A finedgrained exploration at circuit level thanks to NVSim [START_REF] Dong | Nvsim: A circuit-level performance, energy, and area model for emerging nonvolatile memory[END_REF] and at architecture level thanks to gem5 [START_REF] Binkert | The gem5 simulator[END_REF] and McPAT [START_REF] Li | Mcpat: an integrated power, area, and timing modeling framework for multicore and manycore architectures[END_REF] was carried out to achieve the best tradeoff regarding the three metrics: latency, power, area. Compared to other technologies such as Phase-Change Memory (PCM) and Resistive memory (RRAM), STT-MRAM is clearly the most competitive in terms of access latency according to the state of the art [START_REF] Singh Meena | Overview of emerging nonvolatile memory technologies[END_REF]. Moreover, STT-MRAM demonstrates a very high endurance (about 10 15 cycles [START_REF] Huai | PMTJ driven STTMRAMwith 300mm process[END_REF]) which is essential for frequently accessed memories such as caches. Regarding the circuit implementation, this work proposes a hybrid memory by exploiting the inherent architecture of cache with separated tag arrays and data arrays. Hence, the design of such a hybrid cache is more convenient for manufacturing than previous solutions which divide tag arrays and data arrays into different regions to consider different memory technologies [START_REF] Wu | Hybrid cache architecture with disparate memory technologies[END_REF], [START_REF] Wu | Power and performance of read-write aware hybrid caches with non-volatile memories[END_REF].

The rest of the paper is organized as follows: Section II is dedicated to the circuit-level analysis, Section III presents the hybrid cache and its performances, a system-level analysis is presented in Section IV and Section V concludes this paper.

II. CIRCUIT-LEVEL ANALYSIS

This section focuses on the performance analysis of the tag array and the data array of a cache memory considering different associativities and different cache sizes, for a technology node of 32 nm.

The cache line size is fixed at 64 Bytes and a room temperature is considered. This study was performed thanks to NVSim, a performance, energy, and area estimator for Non-Volatile Memories (NVM) which uses an empirical modeling methodology based on the well-known CACTI [START_REF] Muralimanohar | Cacti 6.0: A tool to model large caches[END_REF]. Based on circuit-level data of single bit cell and the desired memory architecture information such as capacity, data width, and type of memory (e.g. Cache, RAM, CAM), NVSim estimates the access time, the access energy, and the total area of a complete NVM chip. This tool also includes optimization settings (e.g. buffer design optimization) and various design constraints to facilitate the design space exploration before the fabrication of the actual NVM chip.

A. Tag array

Tag array is a small, every clock cycle accessed memory containing the upper part of the memory address. This suggest that low read latency and low power consumption are mandatory requirement in an efficient cache organization.

1) 32 kB cache: The memory organization for a small cache size (here 32 kB) was first explored. The associativity impact on the cache sub-array organization is reported in Table I. When doubling the associativity degree, the tag array size increases by around 5%. The same result is predictable using the following relation: Where:

• TagSize is the tag field of the memory address, which increases by 1 bit when the associativity is doubled. • NoSets is the number of sets (i.e. the number of cache line per tag array). • NoWays is the number of ways (i.e. the number of tag arrays). As the number of rows per columns changes, the weight of the peripheral circuits on the overall performance changes too.

Since the tag array is read for each cache access, the latency of this operation is a critical parameter.

• Tag array in SRAM • Tag array in STT-MRAM Fig. 1 shows that for a small size memory, the latency is mainly due to the peripherals circuits.

Because of its small cell size, STT-MRAM based tag array shows a negligible bitline latency. However, it remains slower than a SRAM-based tag array because of the latency of the peripheral circuits, especially the row decoder and the sense amplifiers.

Because STT-MRAM requires large CMOS transistors for the peripheral circuits, this technology is not suitable to build tag array of cache memories according to this analysis.

2) 1 MB cache: A global performance comparison between SRAM and STT-MRAM tag is carried out for a 1MB cache in Figures 2, 3, 4, 5, 6 and 7. Results concerning read and write dynamic power and latency show a clear superiority of SRAM over STT-RAM. Write dynamic energy and write latency are actually the two main drawbacks linked with the physical structure of STT-RAM. They penalize this technology in small cache size.

It is interesting to notice that latency results (Fig. 3 and Fig. 2) of 8-way set associativity for STT-MRAM cache are the highest. This is due to non linearity issues in 8-way set and the impossibility to set some constraints in the sub-array organization in NVSim (such as force the number of banks, mats and sub-arrays as well as number of bit-lines connected to the same multiplexer).

As expected, the main strength of STT-MRAM cells is in leakage consumption as shown in Table II (only the peripheral circuits and access transistors contribute to the static power consumption). Regarding the area (Fig. 6), STT-MRAM tag array overcomes SRAM tag array for every associativity degree, because the bit cells area contribution become more important than the peripheral circuits area contribution for large arrays.

B. Data array

Data array is the part of the cache in which data are stored. As for the tag array analysis, this section explores the impact of the associativity on the data array performance.

• Data array in SRAM:

The overall performance of SRAM-based data array does not change significantly when varying the associativity. Up to 0.5% of variation is observed for the access latency.

The energy consumption remains roughly constant. • Data array in STT-MRAM:

As for SRAM, STT-MRAM based data array keeps the same overall performance by changing the associativity. However, the write dynamic energy decreases linearly with the increase of the associativity, as shown in Table III. In Table III, MuxSenseAmpl represents the number of bitlines connected to each sense amplifier, and Set/reset dynamic energy represent the write dynamic energy per sub-array.

If multiples bit-lines are connected to one sense amplifier, all these bit-lines have to be biased when a single write occurs, that is why the write dynamic energy is inverse correlated to the associativity.

A performance comparison between the two memory technologies considering different cache sizes was explored. Results are summarized in Fig. 7.

To summarize:

• STT-MRAM arrays are better than SRAM ones concerning leakage for every data array size. • STT-MRAM arrays are denser, and so smaller than SRAM arrays. • For small memory sizes, the area is dominated by peripheral circuits, so STT-MRAM is larger than SRAM. • Still for small memory sizes, the latency is dominated by peripheral circuits, so STT-MRAM is slower than SRAM. • When the size is large enough to make the peripheral circuits latency and area negligible compared to the cell array, SRAM memory is slower for read operations and larger than STT-MRAM.

III. HYBRID CACHE

Using the following considerations:

• For small cache size, tag array area and timing performances are dominated by peripheral circuits, and STT-RAM is better than SRAM only for leakage consumption. • For large data array, STT-MRAM is better than SRAM for area, latency and leakage. • Increasing associativity while keeping constant the number of columns and rows, lead to decrease the write dynamic energy. We propose a novel hybrid cache organized as follows:

• Tag array in SRAM • Data array in STT-RAM

A. Experimental setup

Based on the observations of Section II, size and associativity are chosen for their promising results in leakage, area, read latency, read dynamic energy and write dynamic energy: 1 MB, 16-way set associative cache.

Three transistor types were considered:

B. Performances

For each scenario, the write dynamic energy of the hybrid cache is midway between full SRAM and full STT-MRAM write energies (Fig. 9). As expected, a 1 MB cache memory with hybrid banks has worst performances than a full SRAM in write dynamic energy, due to the impact of the STT-MRAM data array. But this is better than for full STT-MRAM, thank to the SRAM tag array. This improvement will also limit the power dissipated in write operations, caused by the required current to flip the spin state.

During a cache block replacement, NVSim writes tag and data in parallel, and so the latency is defined by the worst latency between tag write and data write (here, the data write). This is why the write latency in hybrid banks is always the same as the latency in full STT-MRAM ones (Fig. 10).

Fig. 11 shows that in most of the scenario, the hybrid cache requires lower read energy than SRAM. For the LOP mode with normal access, hybrid bank performances exceeded baseline about 35%. In the LOP mode, electrical SRAM CMOS parameters (Vdd, Vth, Cox...) are modified in order to have a strong cache dynamic energy reduction.

However, the hybrid cache still requires lower energy than full STT-MRAM for read operations.

As expected, the hybrid bank has the best read latency in sequential access mode: in tag array SRAM is faster than STT-MRAM, and in large data array STT-MRAM is the fastest. In the normal access mode, the hybrid bank has the same performances as STT-MRAM banks.

For now, the hybrid cache is at least equal or better than the full STT-MRAM cache.

Fig. 13 shows that the leakage current is greatly decreased when using a STT-MRAM data array; the remaining leakage is due to the peripheral circuits. Even if using a SRAM tag affects this leakage current, the global leakage is still very lower than the leakage of a classic SRAM cache.

Like for the leakage current, Fig. 14 shows that a STT-MRAM data array is better in term of area than a SRAM one. For large cache sizes, using a SRAM tag rather than a STT-MRAM tag does not have a great impact on the area.

To summarize: • Area and leakage: hybrid cache is not as good as full STT-MRAM cache, but still far better than SRAM cache. • Write dynamic energy: hybrid cache are halfway between SRAM and full STT-MRAM caches.

IV. SYSTEM-LEVEL ANALYSIS

In order to go further and analyze the potential gains of a hybrid cache over full SRAM and full STT-MRAM in a realistic scenario, some applications have been simulated using these architectures with the gem5 simulator [START_REF] Binkert | The gem5 simulator[END_REF], a processor architecture simulator, and McPAT [START_REF] Li | Mcpat: an integrated power, area, and timing modeling framework for multicore and manycore architectures[END_REF], an integrated power, area, and timing modeling framework. Details of the simulated architectures are shown in Table IV. Different workloads of the Parsec 3.0 suite have been simulated, and results are summarized in Fig. 15. The timing performance of STT-MRAM over SRAM is really dependent of the application. But, the hybrid cache is always better than a full STT-MRAM caches, as expected. As expected too, the low-leakage of the STT-MRAM has a clear impact on the L2 cache energy consumption, as shown in Fig. 16.

Looking at the global energy consumption in Fig. 17, the cores represent a large part of the energy consumption, and the more cores there are the larger is that part. Still, the L2 cache is the second largest part. It represent early 33% of the global energy consumption in the mono-core architecture for this application. There is clearly a huge advantage to use STT-MRAM in caches in order to reduce the microprocessor consumption.

Finally, there is no relevant differences in global energy consumption using full STT-MRAM or hybrid cache. The gains are dependent of the running application and the architecture, so the main difference is in timing.

V. CONCLUSION AND PERSPECTIVES

A fine-grained tag array and data array study was exposed in this paper and a novel hybrid cache organization was proposed.

Simulations results show that the hybrid cache is a balanced solution which uses both SRAM and STT-MRAM advantages: reducing the energy consumption while maintaining a lowlatency response.

Architecture-level simulations demonstrate that the hybrid cache is indeed better in consumption than a classic SRAM cache, and so we can reduce the global consumption of an architecture using STT-MRAM technology. Also, the hybrid cache is faster than a full STT-MRAM cache, and their impact To go further and enhanced this analysis, a more detailed approach should be done by using parameters of real implementations, in order to obtain a finer comparison and estimation of the gains of the hybrid cache over a full STT-MRAM cache.

TFig. 1 .Fig. 2 .

 12 Fig. 1. Tag sub-array read latency in a 32 kB cache (a) SRAM tag array (b) STT-MRAM tag array

Fig. 3 .Fig. 4 .

 34 Fig. 3. Tag array write latency (1 MB cache)

Fig. 5 .Fig. 6 .

 56 Fig. 5. Tag array write dynamic energy (1 MB cache)

Fig. 7 .

 7 Fig. 7. STT-MRAM data array size and their advantages in NVSim metrics (worst case)

Fig. 8 .

 8 Fig. 8. Hybrid cache

Fig. 10 .

 10 Fig. 10. 1 MB 16-way write latency (relative to classic SRAM)

Fig. 12 .

 12 Fig. 12. 1 MB 16-way cache read latency (relative to classic SRAM)

Fig. 14 .

 14 Fig. 14. 1 MB 16-way cache area (relative to classic SRAM)

Fig. 16 .

 16 Fig. 16. Normalized L2 cache energy, 4 cores at 1 GHz

• High Performance (HP) • Low Operating Power (LOP) • Low Standby Power (LSTP)

• Read latency and dynamic energy: hybrid cache are better than SRAM and full STT-MRAM caches. • Write latency: hybrid cache, like full STT-MRAM cache, are a little slower than SRAM cache.

ACKNOWLEDGMENT This work has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 687973 (GREAT project) and the French National Research Agency under grant ANR-15-CE24-0033-01 (MASTA project).


Sébastien Imbert

Jean-Yves Brossas

Martine Palous

Isabelle Joly

Isabelle Meyer

Arnaud Fekkar
email: arnaud.fekkar@aphp.fr

Performance of Aspergillus PCR in cerebrospinal fluid for the diagnosis of cerebral aspergillosis

Keywords: Aspergillus fumigatus, Aspergillus PCR, Cerebrospinal Fluid, Fungal Infection, Solid Organ Transplant, Haematology, Galactomannan

ABSTRACT.

Objectives: cerebral aspergillosis is a rare but often fatal form of invasive aspergillosis that remains difficult to diagnose. The literature has shown the value of Aspergillus PCR in bloodderived samples for the diagnosis of invasive aspergillosis but provides far less information for cerebrospinal fluid (CSF) in cerebral aspergillosis. Here, we evaluated the usefulness of an Aspergillus PCR assay performed on CSF for the diagnosis of cerebral aspergillosis.

Methods: this retrospective study involved 72 patients with suspected cerebral aspergillosis for a total of 88 CSF samples in whom CSF Aspergillus PCR was performed.

Results: seventeen patients had proven/probable invasive aspergillosis according to the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria, including twelve cases of proven/probable cerebral aspergillosis.

Aspergillus PCR in CSF was positive in nine of the twelve patients with cerebral aspergillosis, i.e. 75% sensitivity. In contrast, CSF culture was positive for Aspergillus in only two patients. In the non cerebral aspergillosis group (60 patients), PCR was positive in one patient, i.e., 98.3% specificity. In this particular population of high-risk patients with suspicion of cerebral aspergillosis, the disease incidence was 16.7%. Therefore, the positive and negative predictive values of PCR were 90% and 95.2% respectively.

Conclusion: the results of this study indicate that

Aspergillus PCR in CSF is an interesting tool that may eliminate the need for cerebral biopsy in patients with suspected cerebral aspergillosis.

INTRODUCTION.

Cerebral aspergillosis (CA) is a rare clinical form of invasive aspergillosis with a mortality rate of up to 90% [1], whose diagnosis remains challenging to this day [2, 3]. Indeed, the diagnostic gold standard relies on histopathological examination of brain tissue biopsies which are rarely performed. Imaging studies, especially magnetic resonance imaging (MRI), can be helpful. Cerebral abscesses and aneurysms are the main characteristics found during CA but these findings are not specific and depend on the mode of dissemination of the infection [4]. Cerebrospinal fluid (CSF) stands as an alternative. It can be subjected to the same tests used in blood-derived samples or in bronchoalveolar lavage samples (BAL) to diagnose invasive aspergillosis, such as culturing, galactomannan determination or PCR.

Several studies have shown the utility of galactomannan determination in CSF for the diagnosis of CA, but few were focused on the performance of Aspergillus PCR despite its demonstrated value for the diagnosis of invasive aspergillosis when performed on bloodderived samples [5,6]. In the few studies that have looked at Aspergillus PCR in CSF, the technique yielded excellent sensitivity and specificity, up to 100% and 93% respectively, but those data need confirmation due to the small number of patients included in the studies [7,[START_REF] Reinwald | Diagnostic performance of an aspergillus-specific nested pcr assay in cerebrospinal fluid samples of immunocompromised patients for detection of central nervous system aspergillosis[END_REF]. Thus, for the present study, we sought to enlarge this knowledge base by evaluating the performance of an in-house A. fumigatus real-time PCR using CSF from patients with suspected cerebral aspergillosis.

MATERIALS AND METHODS.

This retrospective single-centre analysis was performed between February 2012 and January 2016 in La Pitié Salpêtrière hospital, a tertiary care centre in Paris, France. As the study was performed using results get from routine clinical practice, any authorization from a research ethics committee was not required. Patients in whom a CSF Aspergillus PCR was prescribed by clinicians were considered to have a suspected CA and were included in the study. If clinical or radiological data were lacking we thereafter excluded these patients from analyses.

CA was defined as proven when patients had a positive Aspergillus culture in CSF or brain biopsy tissue. It was defined as probable when patients had cerebral radiological findings suggestive of the diagnosis (e.g. cerebral abscess, meningeal enhancement or vasculitis) and a proven or probable invasive aspergillosis elsewhere, as previously reported [START_REF] Chong | Diagnostic performance of galactomannan antigen testing in cerebrospinal fluid[END_REF]. Patients were diagnosed as no CA when they had no invasive aspergillosis, or had invasive aspergillosis with cerebral abnormalities due to another aetiology. We used a real-time PCR that targets a previously-described 67-bp segment of a 28S ribosomal RNA coding DNA [5,[START_REF] Challier | Development of a serum-based taqman real-time PCR assay for diagnosis of invasive aspergillosis[END_REF]. Extraction, amplification and quantification were performed as previously described [START_REF] Imbert | Aspergillus PCR in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients[END_REF], excepted if CSF volume was below 1 mL. In that case, the totality of the CSF sample was centrifuged and DNA extraction was performed on 200 µL of the centrifugation pellet, using the extractions columns of the QIAamp DNA Blood Mini Kit (Qiagen).

RESULTS.

Over the study period, PCR was performed in 86 patients and clinical and radiological data were available for 72 patients (88 CSF samples). Diagnosis of proven or probable CA was made for five and seven patients, respectively. Among them, 8 patients suffered from CA with additional organ involvement and four had only an isolated CA. Finally, 5 patients suffered from proven/probable invasive aspergillosis but without CA (Figure 1). Incidence of CA in this at-risk population was 16.7% (12/72).

Clinical and radiological findings of patients suffering from CA are shown in Table S1.

CSF Aspergillus PCR was positive in nine of the 12 patients with CA, leading to a sensitivity of 75% (95% confidence interval [CI], 62.5-87.5%) (Table 1). Mycological cultures of CSF were positive in only two cases, (sensitivity of 16.7% [95 CI, 0-37.8%]), all for A. fumigatus.

Four biopsy specimens were also positive for A. fumigatus, thus establishing a proven diagnosis. CSF PCR was positive for three of these four biopsy-proven cases and negative for one (this latter having also a negative galactomannan index and a negative mycological culture of CSF).

Of the 60 patients without CA, only one had a positive PCR in CSF and all CSF cultures were negative. Considering these results, the specificity, the positive predictive value and the negative predictive value of the A. fumigatus PCR in CSF were 98.3% (95% CI, 95.5-100% ), 90% (95% CI, 71.5-100%) and 95.2% (95% CI, 89.9-100%) respectively.

Positive and negative likelihood ratio were 45 (95% CI, 6.27-323) and 0.254 (95% CI, 0.10-0.68) respectively, and diagnosis odds ratio was 177 (95% CI, 16-1892).

DISCUSSION.

In this study we found that Aspergillus PCR in CSF is a useful tool for the diagnosis of CA.

In this setting, it provided 75% sensitivity, a rate comparable with its performance in bloodderived samples [5,6]. Specificity, PPV and NPV were very good in our study, respectively 98.3%, 90% and 95.2%.

CA is a rare but severe infection associated with a high mortality rate [1]. Early initiation of antifungal therapy may improve the prognosis, but this is hampered by the diagnosis, which remains a challenge in current practice. Therefore, mycological tools have been proposed for use in CSF. Galactomannan index determination and mycological culture of CSF have been recognised within the EORTC/MSG criteria for the diagnosis of CA [START_REF] De Pauw | Revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (EORTC/MSG) consensus group[END_REF]. Aspergillus PCR, although it has shown its value for the diagnosis of invasive aspergillosis in blood-derived samples, has not yet been included in the EORTC/MSG criteria and expert recommendations for its use carry precautions [START_REF] Patterson | Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the infectious diseases society of america[END_REF]. In our study, CSF PCR greatly outperformed mycological culture, the latter being positive in only two patients (16.7%). Direct evidence of fungal elements in CSF using a silver staining approach was not observed. Although mycological examination of cerebral biopsy tissue was very useful, it was available for only four patients. However, among those four patients, three had positive CSF PCRs, suggesting that PCR could be used as a substitute for particularly invasive and sometimes unattainable brain biopsies. Unfortunately, we were not able to fully compare the performances of PCR to that of the galactomannan index determination because it was determined in only 5 patients.

Because of the small number of cerebral aspergillosis cases, few studies have evaluated the performances of Aspergillus PCR in CSF. Although in this study cases were also limited, we nonetheless report one of the largest series of cerebral aspergillosis cases, and to our knowledge, the largest series with PCR results. Reinwald et al previously reported a sensitivity of 100% for PCR in CSF [START_REF] Reinwald | Diagnostic performance of an aspergillus-specific nested pcr assay in cerebrospinal fluid samples of immunocompromised patients for detection of central nervous system aspergillosis[END_REF]. However, their study comprised only eight cases and employed a nested PCR that can lead to more false-positive results (specificity was 93% in their study) [14]. In our study, the 3 CSF PCR negative results, leading to a comparatively lower sensitivity, may be explained in part by one case of probable CA due to A. niger, a species not recognised by our PCR. The 2 other negative PCR results were found in 2 patients suffering from only cerebral abscesses without associated meningitis (0 and 1 white blood cell in CSF), that can also explain a negative result in CSF.

In conclusion, the results of this study indicate that performing Aspergillus PCR in CSF is an interesting tool for the diagnosis of cerebral aspergillosis and potentially a substitute for invasive cerebral abscess punctures. However, prospective multicentre studies are needed to confirm these results in the largest possible population, and to compare the performance of PCR with that of the galactomannan index when performed on CSF.

TRANSPARENCY DECLARATION.

  The authors declare no conflicts of interest. FUNDING. This work was supported by internal funding.

FIGURE CAPTION Figure 1 :

 CAPTION1 FIGURE CAPTION

  Gauthier L, Joly I, et al. Aspergillus PCR in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients. Clin Microbiol Infect. 2016. 6 Arvanitis M, Ziakas PD, Zacharioudakis IM, Zervou FN, Caliendo AM, Mylonakis E. PCR in diagnosis of invasive aspergillosis: A meta-analysis of diagnostic

	1	Lin SJ, Schranz J, Teutsch SM. Aspergillosis case-fatality rate: Systematic review of
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	2	Kourkoumpetis TK, Desalermos A, Muhammed M, Mylonakis E. Central nervous
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	5	Imbert S,

system aspergillosis: A series of 14 cases from a general hospital and review of 123 cases from the literature. Medicine (Baltimore). 2012; 91: 328-336. 3 Spapen H, Spapen J, Taccone FS, et al. Cerebral aspergillosis in adult critically ill patients: A descriptive report of 10 patients from the aspicu cohort. Int J Antimicrob Agents. 2014; 43: 165-169. 4 Marzolf G, Sabou M, Lannes B, et al. Magnetic resonance imaging of cerebral aspergillosis: Imaging and pathological correlations. PLoS One. 2016; 11: performance. J Clin Microbiol. 2014; 52: 3731-3742.


Edouard Alphandéry
email: edouardalphandery@hotmail.com

Imène Chebbi

François Guyot

Mickaël Durand-Dubief

Use of bacterial magnetosomes in the magnetic hyperthermia treatment of tumors: A review

Use of bacterial magnetosomes in the magnetic hyperthermia treatment of tumours: A review

of magnetosomes needs to be understoodand to measure the risk-benefit ratio, with which to evaluate their use in the magnetic hyperthermia treatment of tumors.

1.

Culture of MTB and preparation of the magnetosomes

Culture of magnetotactic bacteria:

There are several species of MTB that can be cultivated including both marine and freshwater bacteria (MS-1, MV-1, MSR-1 and AMB-1). In this review, we describe the methods used for the growth of the most widely studied and commercially available strains, Magnetospirillum Sp. MSR-1

and AMB-1 MTB (MSR-1 DSMZ-6361 and AMB-1 ATCC-700264). The optimization of the growth of Magnetospirillum gryphiswaldense MSR-1 MTB has been described in a series of three successive publications, [8][START_REF] Liu | Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density[END_REF][START_REF] Zhang | Semicontinuous culture of Magnetospirillum gryphiswaldense MSR-1 cells in an autofermentor by nutrient-balenced and isosmotic feeding strategies[END_REF]. First, MSR-1 MTB were cultivated in growth medium containing magnesium sulfate (MgSO 4 ), sodium thioglocate, yeast extracts, minerals, ferric citrate, ammonium chloride (NH 4 Cl) and sodium lactate in a 42 L fermentor, [8]. This medium was inoculated with 4.2 L of MSR-1 MTB suspension. During growth, the pH was maintained at 7 using fed-batch conditions in which a solution containing sodium lactate, NH [START_REF] Maier-Hauff | Efficacy and safety of intratumoral thermotherapy using magnetic iron-oxide nanoparticles combined with external beam radiotherapy on patients with recurrent glioblastoma multiforme[END_REF] Cl and ferric citrate was added to the growth medium. These conditions enabled maintenance of a sustainable growth rate for the bacteria. The concentration of dissolved oxygen (DO) in the growth medium, which is an important parameter that influences both the growth rate of the bacteria and the magnetosome production yield, was kept below 10% oxygen saturation to enhance the cell biomass. The production of magnetosomes was then triggered by reducing the DO concentration to 1% oxygen saturation. Such low DO values were obtained by progressively increasing the stir rate from 100 rpm up to 300 rpm during the growth of the bacteria, while sterile air was bubbled through the growth medium at a low speed of 0.3 L/min. These conditions allowed the synthesis of a high number of magnetosomes per bacterium (~ 25) and the production of a large percentage of magnetosomes containing cells(between 90 and 100 %). After 60 hours of growth, the magnetosome production yield and the magnetosome productivity for MSR-1 were 42 mg/L and 17 mg/L/day respectively. This growth protocol was further optimized by using a different method for more accurate control of the percentage of oxygen contained within the fermentor, [START_REF] Liu | Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density[END_REF]. This was achieved by changing both the stir rate and the air flow during the growth of the bacteria. After 36 hours of bacterial growth, the magnetosome production yield a magnetosome productivity increased to 83 mg/L and 55 mg/L/day respectively, [START_REF] Liu | Large-scale production of magnetosomes by chemostat culture of Magnetospirillum gryphiswaldense at high cell density[END_REF]. A second improvement was achieved by replacing the sources of carbon and nitrogen in the bacterial growth medium to avoid the accumulation of sodium and chloride ions, which are toxic for non-marine species of MTB such as MSR-1 and inhibit high cell concentrations, [START_REF] Zhang | Semicontinuous culture of Magnetospirillum gryphiswaldense MSR-1 cells in an autofermentor by nutrient-balenced and isosmotic feeding strategies[END_REF]. Under these conditions, after 36 hours of growth, the magnetosome production yield and magnetosome productivity further increased to 168 mg/L and 68 mg/L/day, respectively. Another method used to cultivate MSR-1 MTB, not involving fedbatch conditions, yielded a rapid production of magnetosomes in 4 hours and a magnetosome productivity of 6.3 mg/L/hour and 151/mg/L/day, [START_REF] Heyen | Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor[END_REF]. This method has the advantage of being simpler and therefore more practicable for the industrial production of MTB under good manufacturing practice (GMP) conditions. For the growth of AMB-1 MTB, it has been shown that it is possible to grow these bacteria in a large 1000 liters fermentor which can produce 2.6 g of magnetosomes after 7 days, [START_REF] Matsunaga | Mass culture of magnetic bacteria and their application to flow type immunoassays[END_REF]. In this study, the growth medium was completely saturated with nitrogen gas, [START_REF] Matsunaga | Mass culture of magnetic bacteria and their application to flow type immunoassays[END_REF]. As for MSR-1 MTB, AMB-1 MTB were also cultivated in fed-batch conditions.

These bacteria were grown in a 4 L fermentor in ATCC medium 1653 and maintained at pH 6.75 by adding nitric acid solution. This enabled a magnetosome production yield of 4.5 mg/L, [START_REF] Matsunaga | Enhancement of magnetic particle production by nitrate and succinate fed-batched culture of Magnetospirillum sp[END_REF]. This growth protocol was further optimized by changing the feeding solution and the source of iron, [START_REF] Yang | Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1[END_REF].

AMB-1 MTB were cultivated in a 10 L fermentor with a pH-stat system. The feeding solution contained succinic acid, sodium nitrate, and nitric acid. Iron gallate and iron sulfate were the iron sources found to give the most optmimum yield of magnetosome production. The oxygen concentration was maintained between 9 and 15% in the gas phase, which was found to be the optimum oxygen concentration for magnetosome production. Under these conditions, the magnetosome production yield reached 15 mg/L, [START_REF] Yang | Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1[END_REF].

The optimizations of the growth conditions described above will enable industrial developments, which require the production of a large quantity of magnetosomes. These large volumes can indeed be obtained either by using a growth method that yields high level of magnetosomes via the cultivation of MSR-1 MTB, [START_REF] Zhang | Semicontinuous culture of Magnetospirillum gryphiswaldense MSR-1 cells in an autofermentor by nutrient-balenced and isosmotic feeding strategies[END_REF], or by the culture of MTB in large volumes, [START_REF] Matsunaga | Mass culture of magnetic bacteria and their application to flow type immunoassays[END_REF]. In this regard, iron chelating agents such as hemoglobin, EDTA and rhodamine, could be introduced into the bacterial growth medium to enhance the growth rate of magnetotactic bacteria and to increase the magnetosome production yield, [15]. For example, introducing 0.4 µM of hemoglobin solution into the growth medium of AMB-1 MTB, increased the quantity of magnetosomes produced by a factor of ~ 8, [15]. Finally, reasonably high magnetosome productivity of 6.3 mg/L/hour has been achieved under batch conditions, which are more favorable in an industrial setting than fed-batched conditions, [START_REF] Heyen | Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor[END_REF].

Isolation of the magnetosomes from the magnetotactic bacteria:

There are two reasons why suspensions of MTB could not be used for magnetic hyperthermia treatments of cancers: (i) the concentration of magnetosomes in a suspension of MTB is too low and results in poor heating efficiency and thus absence of any anti-tumor activity, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF], and (ii) health and safety issues preventing the administration of whole MTB into humans. To be used in cancer therapy, the magnetosomes must therefore be isolated from the MTB. The methods used in the preparation of these isolated magnetosomes are described below. To extract the magnetosomes from MTB, four main techniques have been tested, which use sodium hydroxide (NaOH), sonication, a French press or a pressure homogenizer to lyse the bacterial cells. The first tested method involved the mixing of MSR-1 MTB with a 5 M NaOH solution for approximately 16 hours before their centrifugation for 10 hours at 8000 rpm to separate the magnetosomes from the debris of bacterial cells, [START_REF] Philipse | Magnetic colloids from magnetotactic bacteria: chain formation and colloidal stability[END_REF]. This seemed to have resulted in magnetosomes organized in chains, clumps and loops, [START_REF] Philipse | Magnetic colloids from magnetotactic bacteria: chain formation and colloidal stability[END_REF]. The second involved sonications at 300 W for 15 minutes, [START_REF] Sun | Preparation and anti-tumor efficiency evaluation of doxorubicin-loaded bacterial magnetosomes: magnetic nanoparticles as drug carriers isolated from Magnetospirillum gryphiswaldense[END_REF], 80 W for 10 minutes, [START_REF] Taoka | Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum[END_REF], or 30 W for two hours, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF], to lyse MSR-1, MS-1 and AMB-1 MTB, respectively. Using this method, the highest sonication power led to the extraction of aggregated magnetosomes, [START_REF] Sun | Preparation and anti-tumor efficiency evaluation of doxorubicin-loaded bacterial magnetosomes: magnetic nanoparticles as drug carriers isolated from Magnetospirillum gryphiswaldense[END_REF], and the lowest sonication power gave well dispersed chains of magnetosomes, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF][START_REF] Taoka | Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum[END_REF]. The third test, involving the passing of suspensions of MTB two to three times through a French press at a pressure of 1000-20 000 lb/in 2 to lyse AMB-1, [START_REF] Matsunaga | Fully automated immunoassay for detection of prostate-specific antigen nano-magnetic beads and micro-polystyrene bead composites, 'Beads on Beads[END_REF], and MSR-1, [START_REF] Grünberg | Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense[END_REF][START_REF] Xiang | Purified and sterilized magnetosomes from Magnetospirillum gryphiswaldense MSR-1 were not toxic to mouse fibroblasts in vitro[END_REF], MTB resulted in aggregated magnetosomes [START_REF] Grünberg | Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense[END_REF][START_REF] Xiang | Purified and sterilized magnetosomes from Magnetospirillum gryphiswaldense MSR-1 were not toxic to mouse fibroblasts in vitro[END_REF]. Finally, a pressure homogenizer has also been used to lyse MSR-1 MTB, [START_REF] Guo | A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense[END_REF][START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF] at a pressure of 9000-18000 lb/in 2 , [START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF]. While this method does preserve the magnetosome membrane, [START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF], on which magnetosome organization depends , [START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: Sedimentological paelomagnetic, and evolutionary implications[END_REF], only a few transmission electron microscopic images of the magnetosomes isolated using this method have been published making it difficult to confirm with certainty the magnetosome organization, [START_REF] Guo | A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense[END_REF][START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF]. The technique used to lyse the cells, which produces different levels of bacterial cell disruption, does affect the magnetosome organization, [START_REF] Kobayashi | Experimental observation of magnetosome chain collapse in magnetotactic bacteria: Sedimentological paelomagnetic, and evolutionary implications[END_REF].

Purification of the suspension of magnetosomes:

Following extraction of the magnetosomes from whole MTB, several different methods have been suggested for their purification. One of the most comprehensive methods described involved a system of magnetic isolation followed by low power ultrasonication and treatment with urea; treatement with proteinase K to remove adsorbed and surface proteins and with electro-elution to remove nucleic acids, [START_REF] Guo | A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense[END_REF]. The magnetosomes were then lyophilized and sterilized with gamma rays, [START_REF] Guo | A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense[END_REF]. Other techniques have used phenylmethylsulfonyl fluoride to inhibit the activity of the protease; a MACS magnetic column to wash the suspension of magnetosomes with Hepes buffer, [START_REF] Grünberg | A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria[END_REF], and a treatment with 5 µg/ml of DNase I for two hours to remove DNA, [START_REF] Sun | In vitro and in vivo antitumor effects of doxorubicin loaded with bacterial magnetosomes (DBMs) on H22 cells: The magnetic bio-nanoparticles as drug carriers[END_REF]. To examine if the purification was successful, the suspension of magnetosomes was centrifuged and the absorption of the supernatant was measured at 280 nm. Therefore, the amount of biological material remaining in the supernatant could be quantified. A successful purification method would lead to a only a small quantity of biological material remaining in the supernatant and therefore a low absorbance, [START_REF] Gorby | Characterization of the bacterial magnetosome membrane[END_REF].

The percentage of magnetosomes obtained from 1 g of cells depends on the extraction and purification methods used and ranges between 0.6 % and 4 %, [START_REF] Grünberg | Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense[END_REF][START_REF] Grünberg | A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria[END_REF][START_REF] Gorby | Characterization of the bacterial magnetosome membrane[END_REF].

Characterization of the suspension of magnetosomes:

Characterization of the magnetosome suspension is another important aspect to be considered.

Indeed, before administering a suspension of magnetosomes to humans, its full characterization is first required. The biological material surrounding magnetosomes, which is essentially made of fatty acids, phospholipids and proteins, has been mainly characterized using chromatography, infra-red spectroscopy, SDS-page and mass spectroscopy. Previous studies have shown that around 18 proteins were surround the magnetosomes in the MSR-1 species, representing 4% of the total weight, [START_REF] Grünberg | A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria[END_REF]. Lipids represent a higher percentage of the total weight of the magnetosomes, (~ 17%), consisting of 1.4% of neutral lipids and free fatty acids, 5.1% of glycolipids, sulfolipids and phosphatides and 10.5% of phospholipids, [START_REF] Gorby | Characterization of the bacterial magnetosome membrane[END_REF]. The concentration of the magnetosome suspension, which needs to be known before administration, has been estimated previously using optical density at 650 nm, [START_REF] Matsunaga | Fully automated immunoassay for detection of prostate-specific antigen nano-magnetic beads and micro-polystyrene bead composites, 'Beads on Beads[END_REF], or 480 nm, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF], and by a using a destructive assay that measures the iron concentration in the suspension, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. Transmission electron microscopy has been used to measure the sizes of the magnetosome cores, which are made of crystallized iron oxide and range between 20 and 120 nm, [START_REF] Bazylinski | Magnetosome formation in prokaryotes[END_REF]. For AMB-1 and MSR-1 species, the sizes of most the magnetosomes range between 30 and 50 nm and the magnetosomes usually possess a cubooctahedral geometry. The composition of the magnetosome core has been reported to be either magnetite, maghemite or intermediate between magnetite and maghemite depending on the level of exposure of the magnetosomes to oxygen, [START_REF] Alphandéry | Difference between the magnetic properties of the magnetotactic bacteria and those of the extracted chains of magnetosomes: Influence of the distance between the chains of magnetosomes[END_REF]. Magnetic measurements were used to distinguish between these two compositions by measuring the presence (or not) of the Verwey transition, which revealed the presence (or not) of magnetite in the magnetosome core, [START_REF] Alphandéry | Difference between the magnetic properties of the magnetotactic bacteria and those of the extracted chains of magnetosomes: Influence of the distance between the chains of magnetosomes[END_REF]. Cytotoxicity, acute toxicity and immunotoxicity of the bacterial magnetosomes have been assessed

and the magnetosome biodistribution has been studied following both intravenous and intratumoral routes of administration. These studies were carried out using bacterial magnetosomes isolated from MTB and then purified.

Acute toxicity of the magnetosomes:

Despite their bacterial origin, suspensions of magnetosomes have shown little cytotoxic effects on mouse fibroblast, H22, HL60 and EMT-6 cells, [START_REF] Sun | Biocompatibility of bacterial magnetosomes: Acute toxicity, immunotoxicity and cytotoxicity[END_REF]. It was found that 9 µg/mL of bacterial magnetosomes was not toxic for H22, HL60 and EMT-6 cells. For mouse fibroblast cells incubated in the presence of various concentrations of suspensions of bacterial magnetosomes, no cytotoxicity was observed at a magnetosome concentration below 1.3 mg/mL, [START_REF] Xiang | Purified and sterilized magnetosomes from Magnetospirillum gryphiswaldense MSR-1 were not toxic to mouse fibroblasts in vitro[END_REF]. Bacterial magnetosomes incubated with MDA-MB-231 cells did not induce cell death or a significant cell inhibition at a magnetosome concentration below 125 µg/mL, (6).

The acute toxicity of magnetosomes has also been studied in mice and rats. Suspensions of magnetosomes at doses of 270, 360 or 480 mg/kg were injected intravenously. All mice survived except those treated with the highest dose of magnetosomes, i.e. ~ 0.5% of body weight. In comparison, the mice treated with standard chemical SPION began to die after a lower dose of nanoparticles injected at 135 mg/kg, [START_REF] Alphandéry | Chains of cobalt doped magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. In another study, suspensions of magnetosomes were administered to rats intravenously. The rats survived when less than 82 mg/kg of magnetosomes were administered, [START_REF] Sun | Biocompatibility of bacterial magnetosomes: Acute toxicity, immunotoxicity and cytotoxicity[END_REF]. However, for the rats that died in this study (when administered with more than 82 mg/kg of magnetosomes), it was reported that their death may have been accidental, due to bacterial infection and not to the administration of the magnetosome suspension. Histological

Immuno-toxicity of the magnetosomes:

The toxicity of magnetosomes is not likely to be due to iron chemical toxicity since Fe 3 O 4 is relatively insoluble. While dissolved iron ions may be toxic due to a Fenton reaction, which leads to the formation of hydroxyl radicals that are strong oxidants, Fe 3 O 4 does not produce the same toxic effect. Indeed, the magnetosomes are not reported to trigger the Fenton reaction. They even seem to produce the opposite effect by scavenging reactive oxygen species, [START_REF] Guo | Magnetosomes eliminate intracellular reactive oxygen species in Magnetospirillum gryphiswaledense MSR-1[END_REF]. The toxicity of magnetosomes could also be due to their nanometric sizes that might result in embolism, blood clot, cell toxicity or acute toxicity as described above. On the other hand, it could also arise from the magnetosome biological impurities, such as the proteins, the nucleic acids and the lipopolysaccharides (LPS) surrounding the magnetosomes that could lead to potential immunotoxicity, [START_REF]Suspensions magnetosomes were administered intravenously to mice. They were observed in the feces of these mice collected after the administration by transmission electron microcopy[END_REF]. The latter was studied by administering a 1 mg suspension of magnetosomes into the ears of rabbits and then monitoring body temperatures after the injection, [START_REF] Sun | Biocompatibility of bacterial magnetosomes: Acute toxicity, immunotoxicity and cytotoxicity[END_REF]. Body temperature did not increase suggesting the absence of a pyrogenic effect. Moreover, intravenous administration of 40 mg/kg magnetosome suspension into rats revealed no difference in the number of white blood cells and lymphocytes to those in untreated rats. The stimulation index of lymphocyte cells, which is the ratio between the number of proliferating T lymphocytes present in a lymphocyte culture after exposure to antigen to the total number of cells, was also measured for the rats treated (or untreated) with an intravenous administration of a suspension of bacterial magnetosomes using LPS as antigens. No difference was observed in the stimulating index between the treated and untreated rats indicating the absence of immunotoxicity induced by the intravenous injection of 40 mg/kg of bacterial magnetosomes, [START_REF] Sun | Biocompatibility of bacterial magnetosomes: Acute toxicity, immunotoxicity and cytotoxicity[END_REF]. Together, these results indicate that if purified correctly, the magnetosomes do not induce side effects and are of low toxicity. Studies have also been carried out in mice and rats to investigate the biodistribution of magnetosomes following either an intravenous or an intratumoral route of administration. Their aims were to examine the possible degradation of the magnetosomes and to determine the elimination pathways. First studied was the potential decomposition of magnetosomes by lysosomes, known to degrade nanoparticles. For this, a suspension of magnetosomes isolated from MTB was mixed with crude protease from bovine pancreas that simulates intracellular lysosomal conditions, [START_REF] Sun | In vitro and in vivo antitumor effects of doxorubicin loaded with bacterial magnetosomes (DBMs) on H22 cells: The magnetic bio-nanoparticles as drug carriers[END_REF]. The magnetosomes appeared to be degraded after 28 days, suggesting that lysosomes are indeed able to degrade magnetosomes, [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF]. The decomposition of magnetosomes by lysosomes was further confirmed by two experimental results. Firstly, suspensions of bacterial magnetosomes were administered to rats, [START_REF] Sun | Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats[END_REF], or mice, [START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF][START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF], and their localization was determined by fluorescence microscopy, [START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF], or histological analysis of the main organs, [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF][START_REF] Sun | Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats[END_REF], one to fourteen days following administration. Magnetosomes were found in the liver [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF][START_REF] Sun | Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats[END_REF], spleen, [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF], and lungs, [START_REF] Tang | Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice[END_REF], where they were located inside lysosomes in a partly degraded state. In these studies, the magnetosomes that ended up in the liver and spleen were digested by lysosomes, [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF][START_REF] Sun | Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats[END_REF]. Secondly, another study showed that three days following intravenous injection the magnetosomes were neither found in the feces nor in the urine, [START_REF] Sun | Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats[END_REF]. These results seem to indicate that the magnetosomes were degraded by the mice or rats. The hypothesis proposed in light of these results was that the reticuloendothelial system, composed of liver and spleen macrophages, removes magnetosomes from the bloodstream, [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF]. Other studies, however, have found magnetosomes in the feces of mice following both intratumoral and intravenous routes of administrations, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF][START_REF]Suspensions magnetosomes were administered intravenously to mice. They were observed in the feces of these mice collected after the administration by transmission electron microcopy[END_REF]. Taken together, these results suggest that a portion of the bacterial magnetosomes is probably degraded while another portion is eliminated in the feces. Biodistribution studies of chemically synthesized nanoparticles revealed different features.

For example, long-term follow up over a period of 6 months of chemically synthesized nanoparticles injected intravenously showed that these nanoparticles were metabolized in the organ's parenchyma with metal ions leaking from the core material, [START_REF] Lacava | Long-term retention of dextran-coated magnetite nanoparticles in the liver and spleen[END_REF]. However, there are still too few results published on the magnetosome biodistribution to be able to compare in details the biodistribution of the magnetosomes with that of chemically synthesized nanoparticles. Moreover, further studies are necessary to determine more precisely the elimination and degradation pathways of bacterial magnetosomes and to examine whether or not the magnetosomes are metabolized.

Magnetic hyperthermia treatment of tumors with magnetosomes:

3.1 Heating properties of the magnetosomes:

Bacterial magnetosomes are good candidates to carry out magnetic hyperthermia since they produce a large amount of heat when exposed to an alternating magnetic field. In this review, we report values of magnetosome losses per cycle as a measurement of the amount of heat produced by magnetosomes. The magnetosome loss per cycle is defined as the magnetosome specific absorption rate (SAR) divided by the frequency of oscillation of the alternating magnetic field, where the SAR is usually estimated using the formula SAR = ρ s C s (∆T/δt)/C Fe , where ρ s , C s and C Fe are the density, calorific capacity and iron concentration of the sample respectively and ∆T/δt is the slope of the variation in temperature with time estimated at the beginning of the heat treatment, [START_REF] Timko | Magnetic properties and heating Page 81 of 87[END_REF]. Using loss per cycle is convenient since it enables comparison between SAR obtained for different alternating magnetic field frequencies. The production of a large amount of heat by the magnetosomes is due to their large sizes and ferrimagnetic behavior at physiological temperatures. Indeed, since the magnetosomes are monodomain, ferrimagnetic and of cubic shape, the amount of heat that they produce is proportional to their sizes. In addition, due to their thermally stable magnetic moment, the magnetosomes produce a larger amount of heat than the smaller superparamagnetic chemically synthesized nanoparticles. These behaviors have been shown both experimentally and theoretically, [START_REF] Timko | Magnetic properties and heating Page 81 of 87[END_REF][START_REF] Timko | Hyperthermic effect in suspension of magnetosomes prepared by various methods[END_REF][START_REF] Hergt | Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia[END_REF][START_REF] Hergt | Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools[END_REF][START_REF] Dutz | Hysteresis losses of magnetic nanoparticle powders in the single domain size range[END_REF][START_REF] Alphandéry | Heat production by bacterial magnetosomes exposed to an oscillating magnetic field[END_REF][START_REF] Rosensweig | Heating magnetic fluid with alternating magnetic field[END_REF]. For suspensions of magnetosomes exposed to a magnetic field of 6.2 mT, losses per cycles of 0.1-0.2 J/Kg were reported, [START_REF] Timko | Magnetic properties and heating Page 81 of 87[END_REF][START_REF] Timko | Hyperthermic effect in suspension of magnetosomes prepared by various methods[END_REF][START_REF] Hergt | Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia[END_REF][START_REF] Hergt | Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools[END_REF][START_REF] Dutz | Hysteresis losses of magnetic nanoparticle powders in the single domain size range[END_REF]. When the strength of the magnetic field was increased to [START_REF] Matsunaga | Mass culture of magnetic bacteria and their application to flow type immunoassays[END_REF] the losses per cycles increased to 0.5-1 J/Kg [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF][START_REF] Timko | Hyperthermic effect in suspension of magnetosomes prepared by various methods[END_REF][START_REF] Hergt | Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia[END_REF][START_REF] Hergt | Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools[END_REF], showing that the magnetosome loss per cycle strongly increases with increasing magnetic field strength. By comparison with chemically synthesized nanoparticles, these losses per cycle are considerably larger at magnetic field strengths below 10 mT; and either equivalent, [START_REF] Dutz | Hysteresis losses of magnetic nanoparticle powders in the single domain size range[END_REF], or larger at magnetic field strengths above 10 mT, [START_REF] Alphandéry | Heat production by bacterial magnetosomes exposed to an oscillating magnetic field[END_REF]. The heating mechanisms of bacterial magnetosomes have also been studied. Three different types of sample were prepared containing whole MTB, chains of magnetosomes extracted from AMB-1 MTB or individual magnetosomes detached from the chains by heat and SDS treatments, [START_REF] Alphandéry | Heat production by bacterial magnetosomes exposed to an oscillating magnetic field[END_REF]. Figure 1 shows transmission electron microscopy images of whole MTB (Figure 1(a)), chains of magnetosomes extracted from MTB (Figure 1(b)) and individual magnetosomes (Figure 1(c)). When suspensions of whole AMB-1 MTB were mixed with water and exposed to an alternating magnetic field frequency of 108 KHz and strength of 23 to 88 mT, the losses per cycle ranged between 1.1 and 8 J/Kg Fe and the heat released by the magnetosomes was found to mainly arise from the inversion of the magnetosome magnetic moment under the application of an alternating magnetic field (magnetic viscosity). Indeed, it has been demonstrated that the contribution of the rotation of the whole bacteria to the production of heat was negligible, [START_REF] Alphandéry | Heat production by bacterial magnetosomes exposed to an oscillating magnetic field[END_REF]. When the magnetosomes were extracted from whole MTB either as chains or individual nanoparticles, the heat released by the magnetosomes was reported to arise both from the inversion of the magnetosome magnetic moment (magnetic viscosity) and from the magnetosome rotation (hydrodynamic viscous coupling of the medium).

Consequently, under the application of the same magnetic field strengths, the losses per cycle increased considerably to 5-11 J/Kg Fe compared with the values obtained using whole bacteria. It was not possible to determine from this study alone if the magnetosome chains or the individual magnetosomes would be the best candidate to carry out magnetic hyperthermia treatment of tumors since both types of magnetosomes led to high heating efficiencies. The influence on the heating properties of the magnetosome size and magnetosome magnetic anisotropy has also been studied, [15,[START_REF] Alphandéry | Chains of cobalt doped magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. To increase the magnetosome magnetic anisotropy, a 2 µM solution of cobalt 13 quinate was introduced in the bacterial growth medium resulting in an increase in the magnetocristalline anisotropy constant by a factor of ~ 9 from K eff = 12 KJ/m 3 in the absence of cobalt up to K eff = 104 KJ/m 3 in the presence of cobalt quinate, [START_REF] Alphandéry | Chains of cobalt doped magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. This increase in magnetosome magnetic anisotropy resulted in an increase in the SAR, [START_REF] Alphandéry | Chains of cobalt doped magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. To increase magnetosome sizes, iron chelating agents such as EDTA and rhodamine B were introduced in the bacterial growth medium.

This resulted in an increase of the mean magnetosome sizes from 42.5 nm in the absence of iron chelating agents up to 60-65 nm in the presence of rhodamine B or EDTA that produced an enhancement of the magnetosome heating properties, [15].

Antitumor activity of the magnetosomes:

Although bacterial magnetosomes belong to a type of iron oxide nanoparticles that produces a large amount of heat when exposed to an alternating magnetic field, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF][START_REF] Timko | Magnetic properties and heating Page 81 of 87[END_REF][START_REF] Timko | Hyperthermic effect in suspension of magnetosomes prepared by various methods[END_REF][START_REF] Hergt | Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia[END_REF][START_REF] Hergt | Magnetic properties of bacterial magnetosomes as potential diagnostic and therapeutic tools[END_REF][START_REF] Dutz | Hysteresis losses of magnetic nanoparticle powders in the single domain size range[END_REF][START_REF] Alphandéry | Heat production by bacterial magnetosomes exposed to an oscillating magnetic field[END_REF], the anti-tumor activity resulting from a magnetic hyperthermia treatment has to our knowledge only been evaluated in two studies, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF][START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF]. When MCF-7 tumor cells were mixed with bacterial magnetosomes and heated under the application of an alternating magnetic field to reach a temperature of 47 °C, 80% of the cell proliferation was inhibited, [START_REF] Liu | Heating effect and biocompatibility of bacterial magnetosomes as potential materials used in magnetic fluid hyperthermia[END_REF]. It was also mentioned in this study that a temperature of 47 °C inhibited more MCF-7 tumor cells than normal L929 cells. This result further supports the safety of the treatment. Indeed, it suggests that if during the course of a magnetic hyperthermia treatment healthy tissues are heated by mistake, the heat would be less damaging to healthy tissues than to tumor tissues. MDA-MB-231 tumor cells were also incubated with a suspension of magnetosomes and exposed to an alternating magnetic field under various conditions, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. The percentage of cell death and cell inhibition increased with increasing concentrations of magnetosomes, increasing incubation times, magnetic field strength and the number of times that the heat treatment was repeated. This suggests that an efficient treatment can be achieved provided optimum values of these four parameters are chosen. 2(a) shows the set-up used for the treatment using an induction system that generates an alternating magnetic field. The treatments, which used the suspensions of whole MTB, individual magnetosomes and SPION, were inefficient and were unable to stop the growth of the tumor. In contrast, when the heat treatment was carried out using suspensions of chains of magnetosomes, the tumors in several mice disapperaed. . We can conclude from these results that the efficiency of the bacterial magnetosomes is not only due to their large sizes but also comes from their arrangement in chains. When the magnetosomes are organized in chains, they are less prone to aggregation. Indeed, several chains of magnetosomes interact in a head to tail manner in such a way that they form a longer chain. This behavior can clearly be observed in Figure 1. In 15 organization of individual magnetosomes obtained by heat and SDS treatments, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF], or by genetic manipulations of magnetotactic bacteria that led to the removal of the filament binding the magnetosomes together in a chain inside MTB, [START_REF] Scheffel | An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria[END_REF].

We then examined the impact of the organization in chains of the magnetosomes on the heating and distribution uniformity as well as on cellular internalization, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF][START_REF] Alphandéry | Preparation of chains of magnetosomes, isolated from Magnetospirillum magneticum AMB-1 magnetotactic bacteria, yielding efficient treatment of Page[END_REF]. The heating uniformity was studied by infrared measurements and histological analysis, which showed a more uniform heating and distribution throughout the tumor for the chains of magnetosomes than for the individual magnetosomes. The internalization of the chains of magnetosomes inside the cells was observed by two types of measurements. The chains of magnetosomes were mixed with MDA-MB-231 cells and exposed to an alternating magnetic field which resulted in the formation of magnetic cells 5 minutes after the beginning of the treatment. Figure 3(a) shows an eppendorf containing MDA-MB-231 cells mixed with chains of magnetosomes being exposed to the alternating magnetic field. Before the application of the alternating magnetic field, Figure 3(b) shows that the cells are not attracted by a magnet positioned next to the eppendorf and are therefore non-magnetic. Therefore, only a small black spot located next to the magnet is visible corresponding to magnetosomes without cells being attracted by the magnet. By contrast, after the application of the alternating magnetic field, as

shown in Figure 3(c), a large dark spot appeared next to the magnet relating to the magnetic cells attracted by the magnet. MDA-MB-231 cells were also incubated in the presence of the chains of magnetosomes for 1 minute and 24 hours using Prussian blue, which reveals the presence of iron.

Comparison between the chains of magnetosomes and the individual magnetosomes:

Chains of magnetosomes have been defined as assemblies of magnetosomes in which the crystallographic directions of most of the magnetosomes are aligned in the direction of the chain elongation, [44]. Individual magnetosomes are assemblies of magnetosomes in which such alignment does not take place. The chains of magnetosomes differ from the individual magnetosomes by their coating. Using infra-red measurements, it has been shown that the chains of magnetosomes were coated by a mixture of lipids and proteins whereas the individual magnetosomes were only coated by lipids, [START_REF] Alphandéry | Preparation of chains of magnetosomes, isolated from Magnetospirillum magneticum AMB-1 magnetotactic bacteria, yielding efficient treatment of Page[END_REF]. Most probably due to different coatings, the chains of magnetosomes were found to have a zeta potential (-22 mV at pH 7), which differs from that of the individual magnetosomes (+ 10 mV at pH 7), [START_REF] Alphandéry | Preparation of chains of magnetosomes, isolated from Magnetospirillum magneticum AMB-1 magnetotactic bacteria, yielding efficient treatment of Page[END_REF]. The magnetic anisotropy of these two types of magnetosomes was studied by depositing a suspension of these two types of magnetosomes on a substrate under the application of a magnetic field, [45]. Magnetic anisotropy was evaluated by measuring the hysteresis loops of these two types of magnetosomes for a magnetic field applied either parallel or perpendicular to the direction of the alignment and by measuring the difference in coercivity and remanent magnetization between these two field configurations. A larger magnetic anisotropy was found for the chains of magnetosomes than for the individual magnetosomes. This comes from the better alignment of the easy axes in which is larger than that obtained with individual magnetosomes, [START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF].

Conclusion:

In conclusion, we have reviewed the most recent data published on the use of magnetosomes for magnetic hyperthermia treatment of tumors. Studies report that it is possible to produce magnetosomes in large quantity (150 mg/liter/day) and to purify the magnetosomes in such a way that cytotoxicity is avoided. The acute toxicity of the magnetosomes was found to be low with rats surviving up to a quantity of magnetosomes administered of 480 mg per Kg of animal. Suspensions of magnetosomes heated under the application of an alternating magnetic field yield high loss power per cycle of 5-11 J/Kg Fe , suggesting that the magnetosomes are good candidates for magnetic hyperthermia treatments of tumors. Moreover, a proof of concept of a therapy using bacterial

  proof of concept study for the therapy on breast tumors xeno-grafted under the skin of mice,[START_REF] Alphandéry | Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria for application in alternative magnetic field cancer therapy[END_REF]. Four different types of suspensions were tested containing whole MTB (Figure1(a)), magnetosomes organized in chains (Figure 1(b)), individual magnetosomes (Figure 1(c)) and SPION. Each suspension contained 10 mg/mL of iron oxide and 100 µL of each suspension was administered with a syringe into the center of the xeno-grafted breast tumors (~ 100 mm 3 in size) The mice were then exposed three times during twenty minutes to an alternating magnetic field strength of 40 mT and frequency of 198 kHz. The schematic diagram in Figure

  Figure 2(b) shows a mouse during the treatment. Typical temperatures reached during the treatment with the chains of magnetosomes were around 45-46 °C as estimated by infra-red measurements (Figure 2(b)). Thirty days following the treatment, the tumor had disappeared in the mouse treated with the chains of magnetosomes (Figure 2(c)), while it was still located at the skin surface of the mice treated with individual magnetosomes (Figure 2(d)) or SPION (Figure 2(e))

Figure 1 (

 1 Figure 1(a), the chains of magnetosomes contained inside the magnetotactic bacterium are short,

Figures 3 (

 3 Figures 3(d) shows the absence of magnetosome internalization after one minute and an efficient

  chains of magnetosomes than in individual magnetosomes,[45]. The larger magnetic anisotropy of the chains of magnetosomes compared with that of the individual magnetosomes could be another factor responsible for their lower tendency to aggregate. The chains of magnetosomes possess Page 74 of 87 URL: http://mc.manuscriptcentral.com/thth E-mail: dewhi002@mc.medical applications due to their low level aggregation. Such property results in high cellular internalization and in uniform tumor distribution. It makes the chains leave the tumor more rapidly than the individual magnetosomes or chemically synthesized nanoparticles. It also results in a percentage of chains eliminated in the feces following intra-tumoral administration,

Figure 1 :

 1 FIGURES:

Figure 2 :

 2 Figure 2: (a) The set-up used to carry out the treatment of the mice by positioning the mice inside the

Figure 3 :

 3 Figure 3: (a) The set-up used to expose a suspension containing chains of magnetosomes mixed with

Figure 4 :

 4 Figure 4: Micrograph of a tumor tissue collected after the administration of the bacterial

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  main organs (heart, liver, spleen, lung, kidney, brain, intestines, lymph glands and thymus) indicated the absence of hemorrhage, inflammation and hyperemia in the rats treated with less than 82 mg/kg of bacterial magnetosomes.

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