Calcitriol and Punica Granatum Extract Concomitantly Attenuate Cardiomyopathy of Diabetic Mother Rats and Their Neonates via Activation of Raf/MEK/ERK Signalling and Mitigation of Apoptotic Pathways.

We investigated the detrimental effects of diabetes on myocardium of pregestational streptozotocin (STZ)-diabetic mother rats and their neonates via evaluations of oxidative redox, inflammatory and apoptotic pathways, also aiming to characterize whether calcitriol and/or pomegranate peel extract confer myocardial protection in hyperglycaemic dams and their foetuses via modulation of the Raf/MEK/ERK cascade. Sixty Sprague-Dawley female rats were randomized into five groups (N = 12): control, diabetic, diabetic treated with calcitriol and/or pomegranate peel extract (PPE), and mated with non-diabetic healthy males. After confirmation of pregnancy, treatments were kept until gestational day (E-18). Serum and cardiac tissues of mothers and foetuses were collected and processed for biochemical, histopathological, and molecular assessments. We observed that, compared to the control, diabetic mothers showed dramatically increased hyperglycaemia and hyperlipidaemia associated with decreased myocardial functions and disrupted maternal performance. Also, diabetic mothers and their neonates exhibited elevated levels of myocardial injury (troponin I, endothelin 1, creatine kinase-MB, lactate dehydrogenase), with increased pro-inflammatory cytokines (interleukin 1, interleukin 1ß, transforming growth factor ß) and oxidative redox. Concurrently, the MAPK pathway was significantly down-regulated with increased myocardial apoptotic activity. Furthermore, mRNA expression of angiogenic and fibrotic markers was significantly increased. Paradoxically, calcitriol and/or pomegranate peel extract alleviated these diabetic myocardial insults and normalized the aforementioned assayed parameters. Our findings hypothesized that calcitriol and/or pomegranate peel extract exerted cardioameliorative impacts due to their unique anti-oxidative and anti-inflammatory properties, and thus may be a promising treatment that directly targets the secondary myocardial complications of diabetes in dams and their offspring.

Intrinsic nonlinearities in the mechanics of hard sphere suspensions.

The onset of nonlinear responses in near hard sphere suspensions is characterized as a function of oscillatory frequency and strain amplitude. At low frequencies where the viscous behavior dominates, the onset of nonlinearities is driven by increases in rate of strain. At high deformation frequency, where suspension mechanics is dominated by an elastic response, the nonlinear responses occur when deformation exceeds a characteristic strain. This strain is associated with the transient confinement of particles by nearest neighbors and its volume fraction dependence is through cage parameters derived from the high frequency elastic modulus. The onset of nonlinear responses takes on a universal behavior when deformation frequency is normalized by the characteristic time governing the shift from viscous to elastic behavior indicating that this transition is associated with transient particle localization and is expected to be observed for all volume fractions where pair interactions are important.

To do or not to do: axillary nodal evaluation after ACOSOG Z0011 Trial.

Methods of axillary evaluation in invasive breast cancer continue to evolve. The recent American College of Surgeons Oncology Group Z0011 Trial is a prospective, randomized, multicenter trial that compared the survival and locoregional recurrence rates after complete axillary lymph node dissection (ALND) versus sentinel node biopsy (SNB) alone in women with a positive sentinel node in an effort to avoid the complications associated with ALND. As the results of this trial are implemented clinically, affecting surgical management of axillary metastatic disease, radiologists may need to redefine their role in the preoperative assessment of the axilla. Before the Z0011 trial, breast imagers worked to identify axillary metastases preoperatively, allowing appropriate patients to proceed directly to ALND and avoiding the need for SNB. However, the Z0011 trial concluded that ALND may not be necessary in women with metastatic axillary disease who meet the trial criteria. In the Z0011 trial, after 6 years of median follow-up there was no difference in either locoregional recurrence or survival among the women who underwent SNB alone compared with those who underwent ALND, suggesting that ALND is unnecessary in a subset of women with a positive node at SNB. These results raise questions about how aggressively radiologists should pursue percutaneous sampling of axillary nodes, as some practitioners conclude that, in an otherwise eligible woman, positive results from imaging-guided percutaneous biopsy preclude a Z0011 trial-directed pathway. Debate about the best way to implement the results of the Z0011 trial into daily clinical practice exists. It is important for breast imagers to work closely with breast surgeons to provide the most appropriate treatment course for each patient.

Application of Otoform to Study Variations of Sinus Tympani: A Novel Technique.

The sinus tympani is a deep pocket of varying dimensions situated in the retrotympanum. It lies medial to the facial nerve thereby making surgical access difficult. It is this area which is frequently involved in chronic otitis media attico-antral type. Removal of disease from sinus tympani is cumbersome and expedites Cholesteatoma Recidivism. In India, very limited studies have been conducted describing the variations of Sinus tympani. Hence, this study is dedicated to estimating it's variations in volume. This is a one-year Observational Cross-sectional study conducted between January 2020 to December 2020 in Temporal Bone Dissection Lab of Department of Otorhinolaryngology and Head and Neck Surgery, J. N. Medical College, KAHER, Belgaum. 40 Human temporal bone (both left and right) have been dissected. The middle ear cavity was filled carefully with a moulding material i.e. Otoform. Once set, a 3-dimensional model of the middle ear cavity was made, which was removed carefully without causing any damage. The volume of the middle ear cavity was calculated. The other contents and parameters were measured using a measuring probe. Forty bones were dissected. The volume of the sinus tympani was measured to be 8.77 cubic mm with a range of 6.5 cubic mm to. 10.9 cubic mm hence showing that sinus tympani show a larger variability in terms of size, shape and volume. The ponticulus was Complete in 65% of the cases (26 bones) and incomplete in 35% of the cases (14 bones). The ponticulus and subiculum were found to be complete in a well pneumatized mastoid bone. The volume of the middle ear cavity was measured to be 1.17 cubic cm. The sinus tympani volume was calculated in this study employing a novel technique called the Otoform mould in cadaveric temporal bones, enabling for accurate measurement. The study concluded that sinus tympani showed a wide range in volume thus owing that it is the structure which has highest variability in terms of size and shape in the middle ear.

Functional hemodynamic imaging markers for the prediction of pathological outcomes in breast cancer patients treated with neoadjuvant chemotherapy.

Significance: Achieving pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) is a significant predictor of increased likelihood of survival in breast cancer patients. Early prediction of pCR is of high clinical value as it could allow personalized adjustment of treatment regimens in non-responding patients for improved outcomes. Aim: We aim to assess the association between hemoglobin-based functional imaging biomarkers derived from diffuse optical tomography (DOT) and the pathological outcome represented by pCR at different timepoints along the course of NACT. Approach: Twenty-two breast cancer patients undergoing NACT were enrolled in a multimodal DOT and X-ray digital breast tomosynthesis (DBT) imaging study in which their breasts were imaged at different compression levels. Logistic regressions were used to study the associations between DOT-derived imaging markers evaluated after the first and second cycles of chemotherapy, respectively, with pCR status determined after the conclusion of NACT at the time of surgery. Receiver operating characteristic curve analysis was also used to explore the predictive performance of selected DOT-derived markers. Results: Normalized tumor HbT under half compression was significantly lower in the pCR group compared to the non-pCR group after two chemotherapy cycles (p=0.042). In addition, the change in normalized tumor StO2 upon reducing compression from full to half mammographic force was identified as another potential indicator of pCR at an earlier time point, i.e., after the first chemo cycle (p=0.038). Exploratory predictive assessments showed that AUCs using DOT-derived functional imaging markers as predictors reach as high as 0.75 and 0.71, respectively, after the first and second chemo cycle, compared to AUCs of 0.50 and 0.53 using changes in tumor size measured on DBT and MRI. Conclusions: These findings suggest that breast DOT could be used to assist response assessment in women undergoing NACT, a critical but unmet clinical need, and potentially enable personalized adjustments of treatment regimens.

Sequencing-guided design of genetically encoded small RNAs targeting CAG repeats for selective inhibition of mutant huntingtin.

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal, but reduction of wild-type HTT (wtHTT) is undesirable, as it compromises gene function and potential therapeutic efficacy. One promising allele-selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing (RNA-seq) to monitor the processing precision of a limited set of CAG repeat-targeted small RNAs expressed from multiple scaffold contexts. Small RNA-seq identified expression constructs with high-guide strand 5' processing precision and promising allele-selective inhibition of mutHTT. Transcriptome-wide mRNA-seq also identified an allele-selective small RNA with a favorable off-target profile. These results support continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process.

Breast imaging with an ultra-low field MRI scanner: a pilot study.

Breast cancer screening is necessary to reduce mortality due to undetected breast cancer. Current methods have limitations, and as a result many women forego regular screening. Magnetic resonance imaging (MRI) can overcome most of these limitations, but access to conventional MRI is not widely available for routine annual screening. Here, we used an MRI scanner operating at ultra-low field (ULF) to image the left breasts of 11 women (mean age, 35 years ±13 years) in the prone position. Three breast radiologists reviewed the imaging and were able to discern the breast outline and distinguish fibroglandular tissue (FGT) from intramammary adipose tissue. Additionally, the expert readers agreed on their assessment of the breast tissue pattern including fatty, scattered FGT, heterogeneous FGT, and extreme FGT. This preliminary work demonstrates that ULF breast MRI is feasible and may be a potential option for comfortable, widely deployable, and low-cost breast cancer diagnosis and screening.

Comparing Efficiency Epley and Semont Manoeuvre with Videonystagmography.

Videonystagmography (VNG) is useful and reliable in diagnosing vertigo. Benign Paroxysmal Positional Vertigo (BPPV) is the most common peripheral vestibular disorder in adults, and posterior canal is the commonest canalinvolved. The treatment of choice for posterior canal BPPV is repositioning manoeuvres. Epley and Semontmanoeuvres are the two most commonly used treatment manoeuvres for the management of posterior canalBPPV. In this study, we use VNG to compare the two. Epley Repositioning Manoeuvre was found to be moreeffective than Semont Liberatory Manoeuvre. Supplementary Information: The online version contains supplementary material available at 10.1007/s12070-023-03901-3.

Excess cerebellar granule neurons induced by the absence of p75NTR during development elicit social behavior deficits in mice.

Introduction: Recently, the cerebellum has been implicated with non-motor functions, including cognitive and emotional behavior. Anatomical and functional studies demonstrate bidirectional cerebellar connections with brain regions involved in social cognition. Cerebellar developmental abnormalities and injury are often associated with several psychiatric and mental disorders including autism spectrum disorders and anxiety. The cerebellar granule neurons (CGN) are essential for cerebellar function since they provide sensorimotor, proprioceptive, and contextual information to Purkinje cells to modify behavior in different contexts. Therefore, alterations to the CGN population are likely to compromise cerebellar processing and function. Previously we demonstrated that the p75 neurotrophin receptor (p75NTR) was fundamental for the development of the CGN. In the absence of p75NTR, we observed increased proliferation of the granule cell precursors (GCPs), followed by increased GCP migration toward the internal granule layer. The excess granule cells were incorporated into the cerebellar network, inducing alterations in cerebellar circuit processing. Methods: In the present study, we used two conditional mouse lines to specifically delete the expression of p75NTR in CGN. In both mouse lines, deletion of the target gene was under the control of the transcription factor Atoh-1 promotor, however, one of the lines was also tamoxifen-inducible. Results: We observed a loss of p75NTR expression from the GCPs in all cerebellar lobes. Compared to control animals, both mouse lines exhibited a reduced preference for social interactions when presented with a choice to interact with a mouse or an object. Open-field locomotor behavior and operant reward learning were unaffected in both lines. Lack of preference for social novelty and increased anxiety-related behavior was present in mice with constitutive p75NTR deletion; however, these effects were not present in the tamoxifen-inducible mice with p75NTR deletion that more specifically targeted the GCPs. Discussion: Our findings demonstrate that alterations to CGN development by loss of p75NTR alter social behavior, and contribute to the increasing evidence that the cerebellum plays a role in non-motor-related behaviors, including social behavior.

Cortical Brain Injury Causes Retrograde Degeneration of Afferent Basal Forebrain Cholinergic Neurons via the p75NTR.

Traumatic brain injury (TBI) elicits neuronal loss at the site of injury and progressive neuronal loss in the penumbra. However, the consequences of TBI on afferent neurons projecting to the injured tissue from distal locations is unknown. Basal forebrain cholinergic neurons (BFCNs) extend long projections to multiple brain regions including the cortex, regulate many cognitive functions, and are compromised in numerous neurodegenerative disorders. To determine the consequence of cortical injury on these afferent neurons, we used the fluid percussion injury model of traumatic brain injury and assessed the effects on BFCN survival and axon integrity in male and female mice. Survival or death of BF neurons can be regulated by neurotrophins or proneurotrophins, respectively. The injury elicited an induction of proNGF and proBDNF in the cortex and a loss of BFCNs ipsilateral to the injury compared with sham uninjured mice. The p75NTR knock-out mice did not show loss of BFCN neurons, indicating a retrograde degenerative effect of the cortical injury on the afferent BFCNs mediated through p75NTR. In contrast, locus ceruleus neurons, which also project throughout the cortex, were unaffected by the injury, suggesting specificity in retrograde degeneration after cortical TBI. Proneurotrophins (proNTs) provided directly to basal forebrain axons in microfluidic cultures triggered retrograde axonal degeneration and cell death, which did not occur in the absence of p75NTR. This study shows that after traumatic brain injury, proNTs induced in the injured cortex promote BFCN axonal degeneration and retrograde neuron loss through p75NTR.

Immunomodulatory and anti-cancer potential of cloves (Syzygium aromaticum) bud extract and its phytogenic silver nanoparticles.

Clove plant (Syzygium aromaticum) is one of the Myrtaceae family. It's a common flavor in food and the traditional medicine. The study's objective was to ascertain whether the clove bud aqueous extract (CAE) and CAE + nanosilver have any biological effects on immune cells and HT-29 colon cancer cell line. Nanosilver was produced through green synthesis approach using CAE. Produced nanosilver was characterized via electron microscope (scanning, SEM) and ultraviolet-visible spectroscopy. CAE and CAE + nanosilver were examined for their active biomolecules using FTIR analysis, p53 contents using real-time PCR, apoptosis and cell cycle arrest power on HT-29 cancer cell line via flow cytometerty and immunomodulatory potential utilizing MTT assay. Results cleared that a spherical nanosilver with a diameter range of 53 nm was formed by CAE. There were several active biomolecules in CAE and CAE + nanosilver. CAE and CAE + nanosilver increased the p53 protein expression and apoptotic cell number in HT-29 colon cancer cells. CAE and CAE + nanosilver could arrest HT-29 cells at the phase G2/M. CAE and CAE + nanosilver stimulated quiescent and PHA-pre-treated splenic cells at higher concentrations, and CAE suppressed quiescent splenic cell when diluted. In conclusion, the safe edible Syzygium aromaticum plant can be utilized to make anti-tumor agent, essentially for colon tumor. As Syzygium aromaticum plant could stimulate immune cells, it can be used as immune-stimulatory agent that can help fight tumor and tumor development.

MicroRNAs, diet, and cancer: new mechanistic insights on the epigenetic actions of phytochemicals.

There is growing interest in the epigenetic mechanisms that impact human health and disease, including the role of microRNAs (miRNAs). These small (18-25 nucleotide), evolutionarily conserved, non-coding RNA molecules regulate gene expression in a post-transcriptional manner. Several well-orchestered regulatory mechanisms involving miRNAs have been identified, with the potential to target multiple signaling pathways dysregulated in cancer. Since the initial discovery of miRNAs, there has been progress towards therapeutic applications, and several natural and synthetic chemopreventive agents also have been evaluated as modulators of miRNA expression in different cancer types. This review summarizes the most up-to-date information related to miRNA biogenesis, and critically evaluates proposed miRNA regulatory mechanisms in relation to cancer signaling pathways, as well as other epigenetic modifications (DNA methylation patterns, histone marks) and their involvement in drug resistance. We also discuss the mechanisms by which dietary factors regulate miRNA expression, in the context of chemoprevention versus therapy.

Method to improve the localization accuracy and contrast recovery of lesions in separately acquired X-ray and diffuse optical tomographic breast imaging.

Near-infrared diffuse optical tomography (DOT) has the potential to improve the accuracy of breast cancer diagnosis and aid in monitoring the response of breast tumors to chemotherapy by providing hemoglobin-based functional imaging. The use of structural lesion priors derived from clinical breast imaging methods, such as mammography, can improve recovery of tumor optical contrast; however, accurate lesion prior placement is essential to take full advantage of prior-guided DOT image reconstruction. Simultaneous optical and anatomical imaging may not always be possible or desired, which can make the accurate registration of the lesion prior challenging. In this paper, we present a three-step lesion prior scanning approach to facilitate improved accuracy in lesion localization based on the optical contrast quantified by the total hemoglobin concentration (HbT) for non-simultaneous multimodal DOT and digital breast tomosynthesis (DBT) imaging. In three challenging breast cancer patient cases, where no clear optical contrast was present initially, we have demonstrated consistent improvement in the recovered HbT lesion contrast by utilizing this method.

Structural and functional adaptation of the lingual papillae of the Egyptian fruit bat (Rousettus aegyptiacus): specific adaptive feeding strategies.

BACKGROUND: The current investigation was aimed to clarify the correlations between the feeding strategy and lingual structure of the Egyptian fruit bat captured from the Egyptian east desert. MATERIALS AND METHODS: The current work was performed on 12 adult Egyptian fruit bats that were observed grossly and with the help of the stereo, light, and scanning electron microscope. There were three types of the lingual papillae: one mechanical (filiform) and two gustatory (fungiform and circumvallate). RESULTS: There were seven subtypes of filiform papillae recognised on the seven lingual regions. There were few fungiform papillae distributed among the filiform papillae on the lingual tip and two lateral parts of apex and body while fungiform papillae were completely absent in the median part. There were three circumvallate papillae. The central bulb of circumvallate papillae was surrounded by one layer of two segmented circular pad. The lingual tip had cornflower-like and diamond-shaped filiform papillae. CONCLUSIONS: Histochemical results revealed that the lingual glands showed a stronger Alcian Blue (AB)-positive reaction and gave dark blue colour, while the reaction for the periodic acid-Schiff (PAS)-stain was negative. Also, the glands exhibited a blue colour as an indication of positive AB reactivity with combined AB-PAS staining.

Garcinol inhibits cell proliferation and promotes apoptosis in pancreatic adenocarcinoma cells.

Garcinol, or polyisoprenylated benzophenone, isolated from the rind of fruiting bodies of Garcinia indica, has been used in traditional medicine for its potential antiinflammatory and antioxidant properties. The objective of this study was to investigate the effect of garcinol on pancreatic cancer (PaCa) cell viability and proliferation. For this, 2 human PaCa cell lines, BxPC-3 and Panc-1, with wild and mutant k-ras, respectively, were treated with garcinol (0-40 µM). Garcinol significantly (P < 0.05) inhibited cell growth (trypan blue exclusion) by induction of apoptosis in a dose- and time-dependent manner. Flow cytometric analysis revealed G0-G1 phase cell cycle arrest in both cell lines. The molecular mechanism of garcinol's action on PaCa cells was investigated by targeting signaling moieties involved in apoptosis (X-IAP, cIAP, caspase-3, 9, and PARP cleavage), transcription factor NF-κB, believed to contribute toward a chemoresistance phenotype in pancreatic tumors, and molecules associated with neovascularization and metastasis (MMP-9, VEGF, IL-8, and PGE(2)). Garcinol significantly (P < 0.05) augmented antiproliferative, proapoptotic, antimetastatic, and antiangiogenic effects in both PaCa cell types relative to untreated cells. These effects were more pronounced in Panc-1. This is the first report on the therapeutically relevant effect of garcinol in PaCa. Further studies are warranted, based on our findings.

Inter-laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples.

AIMS: To compare the standard culture method with a new, rapid test (ScanVIT-Legionella™) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter-laboratory comparison. METHODS AND RESULTS: All samples were processed by the ScanVIT test according to the manufacturer's instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0.888, P < 0.001). The new test was more accurate in identifying the co-presence of Legionella pneumophila and Leg. non-pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter-laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8.7 vs 16.1%). CONCLUSIONS: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.

Erratum to "Synergistic Effect of Garcinol and Curcumin on Antiproliferative and Apoptotic Activity in Pancreatic Cancer Cells".

[This corrects the article DOI: 10.1155/2012/709739.].

Silver zeolite antimicrobial activity in aluminium heating, ventilation and air conditioning system ducts.

INTRODUCTION: Air pollution in confined environments is a serious health problem, in that most people spend long periods indoors (in homes, offices, classrooms etc.). Some people (children, the elderly, heart disease patients, asthmatic or allergic subjects) are at greater risk because of their conditions of frailty. The growing use of air-conditioning systems in many public and private buildings aggravates this health risk, especially when these systems are not correctly installed or regularly serviced. The aim of our study was to verify the capacity of Ag+ ions to stop the growth of bacteria and moulds inside the ducts of Heating, Ventilation and Air Conditioning system ducts (HVAC) systems when these ducts were lined with active Ag+ ions zeolite-coated panels. MATERIAL AND METHODS: A Y-shaped HVAC model with two branches was used; one branch was made of traditional galvanized iron, as was the whole system, while the other was lined with active Ag+ zeolite-coated polyurethane panels. During the test, samples of dust present inside both ducts were collected and seeded in liquid and solid media to detect bacteria and moulds. The presence of bacteria was also sought in the air emerging from the outlets of both ducts. RESULTS: Tests made on samples of particulate collected from the two different ducts revealed a lower total bacterial load in the samples collected from the Ag+ zeolite-coated duct than in the samples from the traditional Zn galvanized duct. In addition, the values of bacterial load found in the air emerging from the Ag+ ions zeolite-lined duct were 5 times lower than those found in the air from the traditional galvanized iron duct. CONCLUSIONS: The utilization of Ag+ zeolite-coated panels in air-conditioning systems could improve the quality of the emerging air in comparison with traditional installations in galvanized iron. This innovation could prove particularly advantageous in the event of accidents during the installation of air-conditioning systems or of contaminated aerosols coming from outside.

Detection of viable but non-culturable legionella in hospital water network following monochloramine disinfection.

BACKGROUND: Prevention of legionellosis remains a critical issue in healthcare settings where monochloramine (MC) disinfection was recently introduced as an alternative to chlorine dioxide in controlling Legionella spp. contamination of the hospital water network. Continuous treatments with low MC doses in some instances have induced a viable but non-culturable state (VBNC) of Legionella spp. AIM: To investigate the occurrence of such dormant cells during a long period of continuous MC treatment. METHODS: Between November 2010 and April 2015, 162 water and biofilm samples were collected and Legionella spp. isolated in accordance with standard procedures. In sampling sites where MC was <1.5mg/L, VBNC cells were investigated by ethidium monoazide bromide (EMA)-real-time polymerase chain reaction (qPCR) and 'resuscitation' test into Acanthamoeba polyphaga CCAP 1501/18. According to the Health Protection Agency protocol, free-living protozoa were researched in 60 five-litre water samples. FINDINGS: In all, 136 out of 156 (87.2%) of the samples taken from sites previously positive for L. pneumophila ST269 were negative by culture, but only 47 (34.5%) negative by qPCR. Although no positive results were obtained by EMA-qPCR, four out of 22 samples associated with MC concentration of 1.3 ± 0.5mg/L showed VBNC legionella resuscitation. The presence of the amoeba A. polyphaga in the hospital water network was demonstrated. CONCLUSION: Our study is the first report evidencing the emergence of VNBC legionella during a long period of continuous MC treatment of a hospital water network, highlighting the importance of keeping an appropriate and uninterrupted MC dosage to ensure the control of legionella colonization in hospital water supplies.