The Immune Landscape of Cancer.

We performed an extensive immunogenomic analysis of more than 10,000 tumors comprising 33 diverse cancer types by utilizing data compiled by TCGA. Across cancer types, we identified six immune subtypes-wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet, and TGF-ß dominant-characterized by differences in macrophage or lymphocyte signatures, Th1:Th2 cell ratio, extent of intratumoral heterogeneity, aneuploidy, extent of neoantigen load, overall cell proliferation, expression of immunomodulatory genes, and prognosis. Specific driver mutations correlated with lower (CTNNB1, NRAS, or IDH1) or higher (BRAF, TP53, or CASP8) leukocyte levels across all cancers. Multiple control modalities of the intracellular and extracellular networks (transcription, microRNAs, copy number, and epigenetic processes) were involved in tumor-immune cell interactions, both across and within immune subtypes. Our immunogenomics pipeline to characterize these heterogeneous tumors and the resulting data are intended to serve as a resource for future targeted studies to further advance the field.

Restricted myeloperoxidase epitopes drive the adaptive immune response in MPO-ANCA vasculitis.

BACKGROUND: Treatment of autoimmune diseases has relied on broad immunosuppression. Knowledge of specific interactions between human leukocyte antigen (HLA), the autoantigen, and effector immune cells, provides the foundation for antigen-specific therapies. These studies investigated the role of HLA, specific myeloperoxidase (MPO) epitopes, CD4+ T cells, and ANCA specificity in shaping the immune response in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. METHODS: HLA sequence-based typing identified enriched alleles in our patient population (HLA-DPB1*04:01 and HLA-DRB4*01:01), while in silico and in vitro binding studies confirmed binding between HLA and specific MPO epitopes. Class II tetramers with MPO peptides were utilized to detect autoreactive CD4+ T cells. TCR sequencing was performed to determine the clonality of T cell populations. Longitudinal peptide ELISAs assessed the temporal nature of anti-MPO447-461 antibodies. Solvent accessibility combined with chemical modification determined the buried regions of MPO. RESULTS: We identified a restricted region of MPO that was recognized by both CD4+ T cells and ANCA. The autoreactive T cell population contained CD4+CD25intermediateCD45RO+ memory T cells and secreted IL-17A. T cell receptor (TCR) sequencing demonstrated that autoreactive CD4+ T cells had significantly less TCR diversity when compared to naïve and memory T cells, indicating clonal expansion. The anti-MPO447-461 autoantibody response was detectable at onset of disease in some patients and correlated with disease activity in others. This region of MPO that is targeted by both T cells and antibodies is not accessible to solvent or chemical modification, indicating these epitopes are buried. CONCLUSIONS: These observations reveal interactions between restricted MPO epitopes and the adaptive immune system within ANCA vasculitis that may inform new antigen-specific therapies in autoimmune disease while providing insight into immunopathogenesis.

Gain control by layer six in cortical circuits of vision.

After entering the cerebral cortex, sensory information spreads through six different horizontal neuronal layers that are interconnected by vertical axonal projections. It is believed that through these projections layers can influence each other's response to sensory stimuli, but the specific role that each layer has in cortical processing is still poorly understood. Here we show that layer six in the primary visual cortex of the mouse has a crucial role in controlling the gain of visually evoked activity in neurons of the upper layers without changing their tuning to orientation. This gain modulation results from the coordinated action of layer six intracortical projections to superficial layers and deep projections to the thalamus, with a substantial role of the intracortical circuit. This study establishes layer six as a major mediator of cortical gain modulation and suggests that it could be a node through which convergent inputs from several brain areas can regulate the earliest steps of cortical visual processing.

Cholesterol of lipid rafts is a key determinant for entry and post-entry control of porcine rotavirus infection.

BACKGROUND: Lipid rafts are major structural components in plasma membranes that play critical roles in many biological processes including virus infection. However, few reports have described the relationship between lipid rafts and porcine rotavirus (PRV) infection. In this study, we investigated whether or not the locally high concentrations (3-5 fold) of cholesterol present in lipid rafts are required for PRV infection, and further examined which stages of the infection process are most affected. RESULTS: When cellular cholesterol was depleted by methyl-ß-cyclodextrin (MßCD), PRV infectivity significantly declined in a dose-dependent manner. This inhibition was partially reversed upon reintroduction of cholesterol into the system. This was corroborated by the co-localization of PRV with a recombinant, GPI-anchored green fluorescent protein, which functioned as a marker for membranous regions high in cholesterol and indicative of lipid rafts. Changes in virus titer and western blot analyses indicated that depletion of cellular cholesterol with MßCD had no apparent effect on PRV adsorption; however, depletion of cholesterol significantly restricted entry and post-entry of PRV into the cell. Both points of inhibition were restored to near normal levels by the addition of exogenous cholesterol. CONCLUSIONS: We conclude from these studies that membrane-based cholesterol and in particular that localized to lipid rafts, is an indispensable biomolecule for PRV infection, and that cholesterol-based control of the infection process takes place during entry and immediately post-entry into the cell.

Trichinella Nativa Outbreak With Rare Thrombotic Complications Associated With Meat From a Black Bear Hunted in Northern Ontario.

BACKGROUND: Although trichinellosis is known to cause thrombotic disease, serious thrombotic events are rare and have not been previously associated with Trichinella nativa infection. METHODS: Patient interviews and medical chart reviews were conducted on 10 men who became ill following consumption of a common source of black bear meat. Trichinella serology on patient sera as well as polymerase chain reaction (PCR) and larval identification of the meat samples was conducted. RESULTS: All 10 exposed individuals developed an acute illness clinically compatible with trichinellosis, characterized by fever, abdominal pain, and diarrhea, along with eosinophilia ranging from 0.9 × 109/L to 6.1 × 109/L. Within 2 weeks of the diarrheal illness, systemic symptoms developed in all exposed individuals characterized by fever, myalgia, periorbital edema, and fatigue. ST-elevation myocardial infarction and sinus venous tract thrombosis occurred as a complication of trichinellosis in 2 patients. Acute serology was nonreactive in all patients, though convalescent serology was reactive in 6 of 8 (75%) patients for whom sera was available. Multiplex PCR identified T. nativa from the bear meat, and was corroborated by microscopic larval identification. CONCLUSIONS: We report a 100% attack rate of T. nativa from bear meat among those who were exposed, and demonstrate that this species can cause serious thrombotic complications of trichinellosis in humans. Education of hunters and the public regarding the importance of proper preparation of wild game prior to ingestion is warranted.

Long-read sequencing improves assembly of Trichinella genomes 10-fold, revealing substantial synteny between lineages diverged over 7 million years.

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host-pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases.

Parathyroid Adenoma in a Young Female Presenting Multiple Fractures and Postoperative Hungry Bone Syndrome.

A young 18-year-old female patient with general bone pain and history of multiple fractures brought her to our medical attention. Laboratory work showed hypercalcemia and high parathyroid hormone levels in the blood. Radiograph imaging revealed severe scoliosis with multiple vertebrae fractures with decreased bone mineral density. Sestamibi showed parathyroid adenoma. This case emphasizes the importance of maintaining a primary hyperparathyroidism as a differential diagnosis when a young patient presents with a multiple pathologic fractures history.

Improvement of Metabolic Parameters Resulted from Levothyroxine Therapy in Hypothyroid Type 2 Diabetes Mellitus Patient.

This is a case of 51 year-old woman with uncontrolled type 2 diabetes mellitus which was diagnosed 1.5 year earlier, obesity with body mass index 32.2 kg/m2, waist circumference of 113 cm, diffuse goiter with neck circumference 40 cm, and hypertension with blood pressure >140/90 mmHg. Since 6 months ago, she often seemed like having less concentration or daydreaming, chronic fatigue, depression, and low food intake. Her body weight had been increasing over time. Her hypothyroidism clinical scoring Billewicz and Zulewski criterias were 17 and 8 respectively, showing that she tended to experience hypothyroidism. From laboratory examination, fasting blood glucose was 216 mg/dL and 2-hour postprandial blood glucose was 320 mg/dL with level of HbA1c was 9.9%, triglyceride level was 486 mg/dL, HDL cholesterol 46 mg/dL, LDL cholesterol 157 mg/dL, and total cholesterol was 269 mg/dL. Thyroid ultrasound showed a diffuse goiter in both lobes of her thyroid gland.

Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity.

Porcine epidemic diarrhea virus (PEDV) is a pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in newborn piglets. Phage display is a technique with wide application, in particular, the identification of key antigen epitopes for the development of therapeutic and diagnostic reagents and vaccines. To identify antigen epitopes with specificity for PEDV, a monoclonal antibody (MAb-5E12) against the immunodominant region of the PEDV Spike protein (S1) was used as the target for biopanning a 12-mer phage display, random peptide library. After multiple rounds of biopanning and stringent washing, three phage-displayed peptides, designated L, W and H, were identified that recognize MAb-5E12. Sequence analysis showed that the one or more of the peptides exhibited partial sequence similarity to the native S1 sequence 'MQYVYTPTYYML' (designated peptide M) at position 201-212. In combination with software analysis for the prediction of B cell epitopes, aa 201-212 exhibited characteristics of a linear epitope on the PEDV S1 protein. In contrast to peptide M, a consensus motif 'PxxY' was identified on both peptides L and W, and on the S1 protein, but not on peptide H. Peptide M and the MAb-5E12-recognizing peptides L and W significantly inhibited the adsorption of PEDV on the cell surface as monitored through plaque-reduction assays. Furthermore, data from real-time PCR and indirect immunofluorescence assays were consistent with the ability of peptides M, L and W to block viral protein expression and thereby function as antiviral agents for PEDV.

Large enantiomeric excesses in primitive meteorites and the diverse effects of water in cosmochemical evolution.

Carbonaceous chondrites are meteoritic fragments of asteroids that avoided the geological reprocessing of larger planets and allow laboratory probing of early solar-nebula materials. Among these, Renazzo-type (CR) chondrites found in Antarctica appear remarkably pristine and are distinguished by abundant organic materials and water-soluble molecules such as amino acids and ammonia. We present a comprehensive analysis of the organic composition of selected CR meteorites of different petrographic classification and compare compounds' abundance and distribution as they may relate to asteroidal aqueous processing and concomitant evolution of the mineral phases. We found that several CR compounds such as amino acids and sugar alcohols are fully represented in stones with no or minimal water exposure indicating a formation that, if solar, preceded parent body processes. The most pristine CRs also revealed natal enantiomeric excesses (ee) of up to 60%, much larger than ever recorded. However, aqueous alteration appears to affect CR soluble organic composition and abundances, in particular some diastereomeric amino acids may gauge its extent by the consequent racemization of their ee.

Managing Hypertriglyceridemia in Daily Practice.

Hypertriglyceridemia is a form of dyslipidemia, which usually occurs in combination with hypercholesterolemia, high-LDL or low-HDL cholesterol level. Most studies suggest that hypertriglyceridemia is associated with many metabolic disorders such as metabolic syndrome, diabetes, obesity, and also cardio-cerebrovascular diseases. Treatment of hypertriglyceridemia is often not comprehensively addressed by many physicians, who usually only include prescribing drugs without encouraging patients to perform physical activity, to take a true healthy diet for dyslipidemia and to stop smoking. This review article discusses evaluation, diagnosis and a comprehensive, yet simple management of hypertriglyceridemia, which can be easily applied in daily clinical practice.

Recurrent Bilateral Staghorn Stones as a Manifestation of Primary Hyperparathyroidism due to Parathyroid Adenoma.

Primary hyperparathyroidism is a medical condition caused by overactive of parathyroid gland. It is most commonly caused by solitary adenoma of the parathyroid gland. Other causes of this condition are hyperplasia, multiple adenomas, and parathyroid cancer. Primary hyperparathyroidism has some metabolic consequences in the calcium metabolism. Hypercalcemia in patient with primary hyperparathyroidism will resulted to the most important comorbidity that is chronic deposition of calcium in the kidney forming nephrolithiasis or other urolithiasis. It is not uncommon, patient with parathyroid adenoma come to health care professionals with the chief complain of recurrence nephrolithiasis.

Petrography, stable isotope compositions, microRaman spectroscopy, and presolar components of Roberts Massif 04133: A reduced CV3 carbonaceous chondrite.

Here, we report the mineralogy, petrography, C-N-O-stable isotope compositions, degree of disorder of organic matter, and abundances of presolar components of the chondrite Roberts Massif (RBT) 04133 using a coordinated, multitechnique approach. The results of this study are inconsistent with its initial classification as a Renazzo-like carbonaceous chondrite, and strongly support RBT 04133 being a brecciated, reduced petrologic type >3.3 Vigarano-like carbonaceous (CV) chondrite. RBT 04133 shows no evidence for aqueous alteration. However, it is mildly thermally altered (up to approximately 440 °C); which is apparent in its whole-rock C and N isotopic compositions, the degree of disorder of C in insoluble organic matter, low presolar grain abundances, minor element compositions of Fe,Ni metal, chromite compositions and morphologies, and the presence of unequilibrated silicates. Sulfides within type I chondrules from RBT 04133 appear to be pre-accretionary (i.e., did not form via aqueous alteration), providing further evidence that some sulfide minerals formed prior to accretion of the CV chondrite parent body. The thin section studied contains two reduced CV3 lithologies, one of which appears to be more thermally metamorphosed, indicating that RBT 04133, like several other CV chondrites, is a breccia and thus experienced impact processing. Linear foliation of chondrules was not observed implying that RBT 04133 did not experience high velocity impacts that could lead to extensive thermal metamorphism. Presolar silicates are still present in RBT 04133, although presolar SiC grain abundances are very low, indicating that the progressive destruction or modification of presolar SiC grains begins before presolar silicate grains are completely unidentifiable.

Antiviral effect of diammonium glycyrrhizinate on cell infection by porcine parvovirus.

Porcine parvovirus (PPV) can cause reproductive failure in swine, resulting in economic losses to the industry. Antiviral effects of diammonium glycyrrhizinate (DG) have been reported on several animal viruses; however, to date it has yet to be tested on PPV. In this study, the antiviral activity of DG on swine testis (ST) cell infection by PPV was investigated using an empirically determined, non-toxic concentration of DG and three different experimental designs: (1) pre-treatment of virus prior to infection; (2) pre-treatment of cells prior to infection; and (3) direct treatment of virus-infected cells. The results showed that DG possesses potent inhibitory effects on PPV when the virus was treated before incubation with ST cells and that virus infectivity decreased in a dose-dependent manner. Results were confirmed by indirect immunofluorescence assays and real-time quantitative PCR. In addition, deoxycholate was used as a control to exclude the possibility that DG acted as a detergent to inhibit PPV infectivity. The study clearly indicates that DG has a direct anti-PPV effect in vitro.

Trichinella: Becoming a parasite.

Phylogenetic evidence indicates that free-living nematodes gave rise to parasitic nematodes where parasitism evolved independently at least 15 times. The high level of genetic and biological diversity among parasites dictates an equally high level of diversity in the transition to parasitism. We previously hypothesized that horizontal gene transfer (HGT) played an important role in the evolution of parasitism among early ancestors of Trichinella, mediated by an interplay of ecological and evolutionary pathways that contributed to persistence and diversification. We propose that host selection may have been associated with the metabolism of ammonia and engender a new paradigm whereby the reprogrammed nurse cell is capable of generating cyanate thereby enabling the importance of the Trichinella cyanase in the longevity of the cell. Parasites and parasitism have revealed considerable resilience against a backdrop of climate change and environmental perturbation. Here we provide a putative link between key periods in the evolution of Trichinella and major geological and climatological events dating back 500 million years. A useful lens for exploring such ideas, the Stockholm Paradigm, integrates Ecological Fitting (a foundation for host colonization and diversification), the Oscillation Hypothesis (recurring shifts between trends in generalization and specialization relative to host range), the Geographic Mosaic Theory of Coevolution (microevolutionary co-adaptive processes), and the Taxon Pulse Hypothesis (alternating events of biotic expansion i.e., exploitation in evolutionary and ecological time). Here we examine how one or more of these interactive theories, in a phylogenetic-historical context and in conjunction with HGT, may help explain the scope and depth of diversity among Trichinella genotypes.

Order of amino acid recruitment into the genetic code resolved by Last Universal Common Ancestor's protein domains.

The current "consensus" order in which amino acids were added to the genetic code is based on potentially biased criteria such as absence of sulfur-containing amino acids from the Urey-Miller experiment which lacked sulfur. Even if inferred perfectly, abiotic abundance might not reflect abundance in the organisms in which the genetic code evolved. Here, we instead exploit the fact that proteins that emerged prior to the genetic code's completion are likely enriched in early amino acids and depleted in late amino acids. We identify the most ancient protein-coding sequences born prior to the archaeal-bacterial split. Amino acid usage in protein sequences whose ancestors date back to a single homolog in the Last Universal Common Ancestor (LUCA) largely matches the consensus order. However, our findings indicate that metal-binding (cysteine and histidine) and sulfur-containing (cysteine and methionine) amino acids were added to the genetic code much earlier than previously thought. Surprisingly, even more ancient protein sequences - those that had already diversified into multiple distinct copies in LUCA - show a different pattern to single copy LUCA sequences: significantly less depleted in the late amino acids tryptophan and tyrosine, and enriched rather than depleted in phenylalanine. This is compatible with at least some of these sequences predating the current genetic code. Their distinct enrichment patterns thus provide hints about earlier, alternative genetic codes.

Lifestyle and clinical risk factors in relation with the prevalence of diabetes in the Indonesian urban and rural populations: The 2018 Indonesian Basic Health Survey.

Aims: To investigate the differences between Indonesian urban and rural populations in the association of lifestyle and clinical factors with diabetes prevalence. Methods: Using database of the 2018 Indonesian Basic Health Survey, which was conducted in April-May 2018, non-pregnant respondents aged ≥15 years old with available blood glucose data (n urban = 17,129, n rural = 16,585) were included in this study. The diagnosis of diabetes was based on the combination of known diabetes, i.e., a previous history of diabetes or use of anti-diabetes medication, and unknown diabetes based on blood glucose criteria. We performed logistic regression analyses separately for the urban and rural populations to examine the association of lifestyle and clinical factors with prevalent diabetes. Results: Indonesian urban population was less physically active, had a lower proportion of adequate fruit and vegetable intake, and had higher individuals with obesity than rural population. Although there were no differences in the total prevalence of diabetes between the two populations (10.9 % vs. 11.0 %, for urban and rural, respectively), the prevalence of known diabetes was twice higher in urban than in rural population (3.8 % vs. 1.9 %). Physical activity was associated with lower risk of diabetes, especially in the urban population [prevalence OR (95 %CI): 0.91 (0.85; 0.98) for urban and 0.94 (0.89; 1.00) for rural). Obesity, hypertension, and dyslipidemia were risk factors for prevalent diabetes in both populations. Conclusions: Indonesian rural population showed relatively better lifestyle and clinical profiles compared to their urban counterparts. However, no differences were observed between the two populations in the relation between risk factors and diabetes. Special attention needs to be addressed to the high prevalence of undiagnosed and untreated diabetes in Indonesia.

*Inactivation of encysted muscle larvae of Trichinella spiralis in pigs using Mebendazole.

We evaluated the effect of 4 anthelmintic treatments on the viability of Trichinella spiralis encysted muscle larvae (ML) 55 days post infection (PI) in experimentally infected pigs. Muscle larvae were isolated from pig muscle by artificial digestion after oral treatment of pigs with Levamisole (8 mg/kg, daily for 5 days) and Mebendazole (50 mg/kg, daily for 5 days); Doramectin (0.3 mg/kg, single IM injection), and Moxidectin (0.5 mg/kg, single pour on). Isolated larvae from treated pigs were orally inoculated into mice to assess viability of ML from each treatment. Only Mebendazole treatment of pigs significantly reduced ML viability in mice. The effect of timing of the effective Mebendazole treatment on ML from a longer term infection was then examined in a second experiment. Analysis revealed that Mebendazole treatment of pigs with 250 mg/kg over 3 days (83 mg/kg/day) or 5 days (50 mg/kg/day) reduced numbers of ML recovered from pig tissues compared to untreated, infected controls, and rendered ML non-infective to mice; Mebendazole treatment of pigs with 250 mg/kg in a single dose was not effective in reducing ML numbers recovered from pigs or in impacting ML infectivity to mice. An examination of the lowest effective dose of Mebendazole on encysted ML was determined in a third experiment. Mebendazole of pigs with 5, 50, or 100 mg/kg over 3 days demonstrated that 5 or 50 mg/kg over 3 days insufficient to reduce infectivity in recovered ML, while 100 mg/kg (and 83 g from experiment 2) over 3 days significantly reduces infectivity of ML. This procedure provides a means to evaluate the efficacy of various anthelmintic treatments on the viability of Trichinella spiralis ML in pig tissues, and identified Mebendazole, at 83-100 mg/kg administered over a 3-5 day period as an anthelmintic which renders encysted Trichinella spiralis ML from pig tissues non-infective. As risk from Trichinella significantly impacts acceptance of pork from pasture-raised pigs, these data provide a method, especially for producers of these high-risk pigs, to eliminate the potential of Trichinella transmission from infected pork.

Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.