The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a respiratory pathogen with remarkable properties of multisystem involvement and numerous complications. The coronavirus disease of 2019 (COVID-19) presenting as stroke is becoming more common. Herein, we describe an unusual case of COVID-19 in a patient who initially presented with myalgia, fatigue, loss of taste and smell, and non-specific memory impairment. Two months after infection with SARS-CoV-2, the patient presented with acute onset of right-sided weakness, sensory loss, and worsening cognitive impairment. Reverse transcription-polymerase chain reaction was performed to detect the SARS-CoV-2 virus, and the results were positive at the time of initial infection as well as during the current admission. Neuroimaging suggested a subacute ischemic infarct in the middle cerebral artery. The patient was re-tested for SARS-CoV-2 and found to be positive, but the cycle threshold was high (40.4) along with a positive test for immunoglobulin-G (IgG) for SARS-CoV-2. This report highlights a unique case of stroke-related to COVID-19 infection in a middle-aged woman with otherwise mild symptomatic illness. The patient had a chronic ischemic stroke with delayed presentation two months after the initial symptomatic viral infection. This case underscores the importance of neurological assessment as well as timely evaluation of patients presenting with COVID-19 and any neurological concerns to recognize stroke as a complication of COVID-19 promptly. Novel coronavirus disease 2019 (COVID-19) is associated with significant morbidity and mortality, with more than 19 million cases and 727,000 deaths worldwide as of August 8, 2020 -7. The iA 59-year-old white woman presented to the emergency department with acute right-sided weakness and memory loss. Six hours before the onset of her weakness, she went to sleep, and upon waking in the morning, she felt weakness in the right upper and lower extremities associated with tingling and numbness in her right foot. She also had an associated mild headache that began at approximately the same time after waking and was located mainly behind her right eye. She was experiencing memory deficits, such as difficulty remembering the names of her children and her work schedule for the day, as well as the names of supermarkets near her house, as noted by her family member. She was diagnosed with COVID-19 two months prior to the current admission when her symptoms included myalgia, loss of taste and smell, and fatigue for one week. She was not hospitalized at that time, and after viral detection, she observed home quarantine for two weeks. She reported feeling mental fogginess and experienced difficulty with memory and executive functioning after the viral illness. However, her memory deficits acutely worsened on the morning of presentation, and the right-sided weakness was new for her. She worked as a radiology technician at a local hospital. Her past medical history was significant for hypertension controlled on losartan and osteoarthritis, for which she took occasional ibuprofen. There was no history of stroke or any other neurological concerns in the past. She denied any history of blood clots, heart disease, or any features of autoimmune disease in the past. She underwent knee replacement for arthritis two years ago. She denied any history of smoking, alcohol, or any illicit drug use. Her family history was significant for Parkinson’s disease, which afflicted her mother. On physical exam, she was afebrile, her heart rate was 78 beats per minute, blood pressure was 140/80 mmHg, and respiratory rate was 18/minute. Her body mass index was 30.1 kg/m2. She was awake, alert, and oriented to time, place, and person. Her speech was fluent but non-purposeful with confabulation. She was displaying some evidence of word salad. When asked a question, she would also often sputter and go off on tangents, and it was hard to redirect her. The results of her cranial nerve exam were unremarkable except her visual field exam suggested superior quadrantanopia on the right side. Her motor exam showed 3/5 weakness on her right side in the lower extremity and normal strength elsewhere. Her sensory examination revealed diminished light touch in the dorsum of the right foot with pinprick, and vibration intact bilaterally. All the deep tendon reflexes were intact, and Babinski’s reflex was negative. Based on her history and exam findings, her National Institute of Health Stroke Scale score was 4 . Her nasopharyngeal swab tested positive for novel SARS-CoV-2 reverse transcriptase-polymerase chain reaction (RT-PCR). Other laboratory findings were suggestive of lymphopenia, with an absolute lymphocyte count of 800 cells/mm3. The serum immunoglobulin-G (IgG) antibody test for SARS-CoV-2 was positive. The inflammatory markers were within normal range and are shown in Table Computed tomography (CT) of the head without contrast showed left frontal lobe hypodensity, as shown in Figure We compared the current CT scan to a previous CT scan of her head (which was done two years ago) that showed no lesion and was reported normal. CT angiography of the head, brain, and neck with contrast revealed minimal calcification at the left common carotid bifurcation without stenosis. The mid-cervical segment of the bilateral internal carotid arteries was tortuous Figure .There was mild diffuse narrowing in the distal M1 and M2 segment of the left middle cerebral artery (MCA). There was no proximal branch occlusion or aneurysm. CT of the head/brain with perfusion (CTP) indicated matched perfusion defect in the left frontal lobe, suggesting core infarct. There was no definitive evidence of any mismatch perfusion defect to suggest reversible ischemic penumbra. Magnetic resonance imaging of the brain with contrast revealed hyperintense signal abnormalities in the left frontal lobe involving the operculum and insula, which were suggestive of an old infarct in the left MCA territory. There was mild ex vacuo dilatation of the left lateral ventricle Figure , 2B, 2C.The patient did not qualify for tissue plasminogen activator administration, considering the delay in presentation and the time frame allotted for it in the case of ischemic stroke . With thWe present an interesting case of COVID-19 infection in a middle-aged woman presenting with stroke, manifesting as a cognitive impairment that was undetected during her initial COVID-19 infection two months earlier. The patient exhibited a mild symptomatic viral illness with non-specific neurological complaints such as loss of olfaction, taste, and fatigue, but began having some degree of memory impairment without any other neurological symptoms. However, she presented two months later with worsening cognitive impairment, as well as sensory deficits and new right-sided weakness. Apart from hypertension, which was controlled by medication, she did not have any underlying vascular risk factors for stroke. COVID-19 infection is known to increase the risk of stroke and various thromboembolic complications . Initial reports suggest that COVID-19 can present with new-onset neurological impairment and even massive stroke, especially in young patients. The majority of neurological complications, especially stroke, occurred in patients who were critically ill ,7. HyperNasopharyngeal swab tests indicated that the patient continued to test positive for SARS-CoV-2 two months after the initial test. Viral shedding with continued positive RT-PCR results for SARS-CoV-2 has been reported at eight weeks, even in patients with mild illness ,16. RepeInterestingly, the neuroimaging was suggestive of an old infarct with new-onset symptoms, which may possibly be related to post-stroke recrudescence (PSR). This has been previously very well described in a cohort study that included 153 patients, of whom 112 patients (73%) exhibited re-emergence of previous stroke-related deficits involving white matter tracts and MCA territory . Stress,This report highlights a unique case of late-onset stroke symptoms related to SARS-CoV-2 infection. The patient had a chronic ischemic stroke in the setting of a recent SARS-CoV-2 infection manifesting as memory impairment. The acute neurological deficits two months after infection may be related to stroke recrudescence during the recovery from SARS-CoV-2 infection. This case elucidates the importance of neurological assessment as well as timely evaluation of patients presenting with COVID-19 and any neurological complaints. Further studies are required to determine the risk factors, pathophysiology, treatment, and prevention modalities for acute strokes related to SARS-CoV-2 infection.


Wearing masks is a CDC-recommendedThe Kansas mandate requiring the wearing of face coverings in public spaces became effective July 3, 2020. Data on county mask mandates were obtained from the Kansas Health Institute.Daily county-level COVID-19 incidence  was calculated using case and population counts accessed from USAFactsAs of August 11, 24 (23%) Kansas counties had a mask mandate in place, and 81 did not. Mandated counties accounted for two thirds of the Kansas population Trend analyses using segmented regression  indicateAfter implementation of mask mandates in 24 Kansas counties, the increasing trend in COVID-19 incidence reversed. Although rates were considerably higher in mandated counties than in nonmandated counties by the executive order, rates in mandated counties declined markedly after July 3, compared with those in nonmandated counties. Kansas counties that had mask mandates in place appear to have mitigated the transmission of COVID-19, whereas counties that did not have mask mandates continued to experience increases in cases.The findings in this report are consistent with declines in COVID-19 cases observed in 15 states and the District of Columbia, which mandated masks, compared with states that did not have mask mandates  within counties before and after July 3. Nonetheless, in additional analyses examining testing data among Kansas counties during the study period, testing rates were observed to increase overall over time. Therefore, despite increases in testing during this period, decreases in COVID-19 incidence were observed in mandated counties after July 3. Finally, counties in Kansas with a mask mandate might not be representative of other U.S. counties. However, the findings are consistent with observations from other states that mask mandates are associated with declines in COVID-19 cases (Masks are an important intervention for mitigating the transmission of SARS-CoV-2 (Wearing face masks in public spaces reduces the spread of SARS-CoV-2.The governor of Kansas issued an executive order requiring wearing masks in public spaces, effective July 3, 2020, which was subject to county authority to opt out. After July 3, COVID-19 incidence decreased in 24 counties with mask mandates but continued to increase in 81 counties without mask mandates.Countywide mask mandates appear to have contributed to the mitigation of COVID-19 transmission in mandated counties. Community-level mitigation strategies emphasizing use of masks, physical distancing, staying at home when ill, and enhanced hygiene practices can help reduce the transmission of SARS-CoV-2.


Background: Aging is characterized by adipose tissue senescence, inflammation, and fibrosis, with trunk fat accumulation. Aging HIV-infected patients have a higher risk of trunk fat accumulation than uninfected individuals—suggesting that viral infection has a role in adipose tissue aging. We previously demonstrated that HIV/SIV infection and the Tat and Nef viral proteins were responsible for adipose tissue fibrosis and impaired adipogenesis. We hypothesized that SIV/HIV infection and viral proteins could induce adipose tissue senescence and thus lead to adipocyte dysfunctions. Methods: Features of tissue senescence were evaluated in subcutaneous and visceral adipose tissues of SIV-infected macaques and in human adipose stem cells (ASCs) exposed to Tat or Nef for up to 30 days. Results: p16 expression and p53 activation were higher in adipose tissue of SIV-infected macaques than in control macaques, indicating adipose tissue senescence. Tat and Nef induced higher senescence in ASCs, characterized by higher levels of senescence-associated beta-galactosidase activity, p16 expression, and p53 activation vs. control cells. Treatment with Tat and Nef also induced oxidative stress and mitochondrial dysfunction. Prevention of oxidative stress (using N-acetyl-cysteine) reduced senescence in ASCs. Adipocytes having differentiated from Nef-treated ASCs displayed alterations in adipogenesis with lower levels of triglyceride accumulation and adipocyte marker expression and secretion, and insulin resistance. Conclusion: HIV/SIV promotes adipose tissue senescence, which in turn may alter adipocyte function and contribute to insulin resistance. Aging is a major issue in the general population; it is associated with adipose tissue senescence, inflammation, and fibrosis, leading to trunk fat redistribution ,2,3. It Given that highly effective antiretroviral therapy (ART) can control but not cure HIV infections, HIV-infected patients are aging with the virus. Moreover, the prevalence of comorbidities typically associated with aging  is higher in people living with HIV that in the general population. In particular, the risk of trunk fat accumulation (associated with pro-atherogenic metabolic factors) is greater in aging HIV-infected people than in uninfected controls, and this risk increases with age ,4,5. TheCellular aging  is defined as an irreversible growth arrest  and an iIn the context of HIV infection, the results of previous in vitro studies have shown that some ARTs (such as protease inhibitors and nucleoside reverse transcriptase inhibitors) can induce senescence in human and murine mesenchymal stem cells (MSCs) ,16,17. E+ T cells and macrophages [Subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) are known to be viral reservoirs that notably contain HIV-infected CD4rophages ,21. Thusrophages . Taken aTherefore, the objective of the present study of two complementary in vivo and in vitro models was to evaluate the impact of HIV/SIV on adipose tissue aging, inflammation, and oxidative stress. First, we revealed the impact of the virus per se on adipose tissue aging in vivo in a novel nonhuman primate model of chronic SIV infection. Then, we showed that the HIV proteins Tat and Nef released by infected cells can induce senescence in human ASCs in vitro—probably through the induction of oxidative stress. Nef-induced senescence impacted the adipogenic capacity of ASCs and resulted in insulin resistance—suggesting that adipose tissue senescence contributes to the fat alterations and cardiometabolic complications observed in HIV-infected people.Macaca fascicularis) were imported from Mauritius, handled, and housed in the animal facilities at the Commissariat à l’Energie Atomique et aux Energies Alternatives . The CEA animal facilities comply with the Standards for Human Care and Use of Laboratory of the Office for Laboratory Animal Welfare  and with the European Directive . The study was authorized by the local animal care and use committee  and the French Ministry of Research (Ministère de l’Enseignement Supérieur et de la Recherche). Macaques were intravenously infected  with SIVmac251, as described previously [4 ± 3.2 × 104 RNA copies/mL. SIV-infected macaques  were compared with noninfected controls  housed under similar conditions.Adult cynomolgus macaques (2) by liposuction during plastic surgery. Donors were nonobese and nondiabetic and had no history of HIV, HCV, or HBV infection. Briefly, SCAT samples were digested with collagenase, filtered to remove large debris, and cultured at an initial density of 5 × 104 cells/cm2 in alpha-minimum essential medium (αMEM) supplemented with 10% fetal bovine serum , 2 mmol/L glutamine, 2.5 ng/mL basic fibroblast growth factor , and penicillin/streptomycin , as described previously [N-acetyl-cysteine (NAC) : After 15 days of treatment with HIV proteins, we started a concomitant NAC treatment for 10 days. Thus, the impact of NAC was analyzed at day 25.The experimental procedures involving human ASCs were approved by the local Ethical Committees for human research and were performed in accordance with the European Union’s guidelines and the Declaration of Helsinki. ASCs were isolated from human abdominal subcutaneous adipose tissue (SCAT) from nine healthy women  fASC proliferation was evaluated in terms of the cell population doubling level , where After 30 days of treatment with Tat or Nef, differentiation of the ASCs into adipocytes was triggered for 14 days in the absence of HIV proteins. ASCs were cultured for six days in adipogenic induction medium  and then cultured in adipogenic maintenance medium (1 µM insulin). On day 14, cells were stained with Oil-Red-O (Sigma-Aldrich), as described previously . The Oild-galactopyranoside  was used as a biomarker of cellular senescence. To quantify SA-β-galactosidase activity, cells were incubated with appropriate buffer solution containing X-Gal at pH 6, as described previously [The blue staining produced by SA-β-galactosidase’s hydrolysis of 5-bromo-4-chloro-3-indolyl-β-eviously . The proThe levels of human IL-6 and IL-8 secreted by ASCs after 15 days of incubation with Tat or Nef were determined in the culture medium from the last 24 h, using Quantikine sandwich ELISAs . The assay sensitivity was 0.7 pg/mL for IL-6 and 3.5 pg/mL for IL-8. Total levels of human adiponectin/Acrp30 and leptin secreted by differentiated ASCs after 14 days of differentiation were determined in the culture medium from the last 24 h, using Quantikine sandwich ELISAs (R&D Systems). The assay sensitivity was 0.246 ng/mL for total adiponectin/Acrp30 and 7.8 pg/mL for leptin.2DCFDA, Invitrogen Corporation) on a plate fluorescence reader at 520–595 nm. Results were normalized against DAPI fluorescence, as described previously [The production of reactive oxygen species (ROS) was assayed by quantifying the oxidation of 5-6-chloromethyl-2,7-dichlorodihydro-fluorescein diacetate  and mRNA expression was analyzed using RT-PCR, as described previously . The seqProteins were extracted from the VAT and SCAT of macaques and ASC monolayers, as described previously , and theExperiments were performed at least three times on triplicate samples. Data are expressed as the mean ± SEM. Statistical significance for HIV-protein-treated cells vs. control cells with or without NAC, and for SIV-infected vs. control macaques were determined with a parametric test (Student’s t-test) or a nonparametric test (the Mann–Whitney U-test), as appropriate. We also performed a two-way ANOVA to study the impact of adipose tissue localization on senescence markers in our in vivo model.p = 0.0009) and of adipose tissue localization (p = 0.05) for p16 expression. Thus, according to our results, the greater expression of p16 in VAT, suggests that VAT displays a higher aging phenotype. Moreover, p16 level and p53 activation in SCAT or VAT did not correlate with viral load, suggesting that the level of senescence was not linked to the severity of SIV infection. These results indicate that adipose tissue was more senescent in infected macaques and strongly suggest that SIV per se accentuates the aging of adipose tissue.We observed that VAT and SCAT p16 expression was higher in infected macaques than in control macaques A. FurtheNext, we looked at whether the HIV proteins Tat and Nef could induce senescence in ASCs. To this end, we first determined the impact of up to 30 days of exposure to Tat or Nef on cell proliferation in vitro. We found that Tat and Nef lowered the ASCs proliferation rate. This effect was seen after 15 days, and the low proliferation rate fell further with each cell passage A, when cROS production (measured through NBT or CM-H2-DCFDA oxidation) was elevated in Tat- or Nef-treated ASCs A. Using 2-DCFDA oxidation  for further 10 days. Experiments were performed on day 25. NAC suppressed the elevation in ROS production induced by Tat or Nef, as shown by NBT and CM-Hxidation A. NAC alxidation B. Accordxidation C and thexidation D. The saPPARG, CEBPA, and FABP4  resulted in a lower level of lipid accumulation A and lownd FABP4 B. Furthend FABP4 C.Taken as a whole, adipogenic fate was affected in ASCs presenting Nef-induced senescence. The absence of an effect on Tat-treated ASCs in this setting might have been due to the protein’s weaker ability to induce senescence, inflammation, and oxidative stress.In order to assess the functional status of adipocytes derived from Tat- or Nef-treated ASCs, we evaluated the cells’ insulin sensitivity. A Western blot analysis showed that Tat- and Nef-treated cells expressed similar, normal levels of total Akt the key enzyme in the insulin signaling pathway, and mainly involved in short-term metabolic responses A. DespitOur present results showed that SIV/HIV infection was associated with certain features of adipose tissue aging. Firstly, fat from SIV-infected macaques displayed markers of senescence. Since adipose tissue has been identified as an HIV reservoir that contains infected cells , To the best of our knowledge, the level of senescence in the adipose tissue of SIV-infected macaques or in ART-naïve, HIV-infected people has not previously been evaluated. Here, we showed that SIV infection is associated with higher adipose tissue levels of p16 in VAT and SCAT and activated p53 in SCAT , which Adipose tissue is an HIV/SIV reservoir even in the presence of suppressive ART ,21,27. FWith regard to the mechanisms that might promote senescence in ASCs, greater mitochondrial dysfunction and ROS production have previously been linked to the occurrence of age-dependent senescence in ASCs from individuals not infected with HIV . HoweverWe observed that Tat and Nef induced a pro-inflammatory profile in ASCs. The profile might contribute to inflammaging, i.e., a low-grade, chronic, systemic inflammatory state that can be induced by oxidative stress or by pathogens. In turn, inflammaging might enhance the aging of adipose tissue. Moreover, inflammation can trigger senescence and promote fibrosis (collagen production) ,40. We hOne of the main characteristics of aging is a decline in the tissues’ regenerative potential. Adipose tissue is a source of multipotent stem cells, namely ASCs ,42. TherPPARG, CEBPA, and FABP4), adipokine secretion, and insulin sensitivity. By removing Tat or Nef during ASC differentiation, we ruled out a direct effect of these HIV proteins on adipogenesis. Thus, our data suggest that the senescence and dysfunction induced by HIV proteins during ASC proliferation partially suppressed adipogenesis. The present results are in line with our previous work, in which we showed that HIV/SIV infection was associated with the presence of smaller adipocytes in both SIV-infected macaques and HIV-infected people [It is known that senescence of adipose tissue precursor cells is associated with low adipogenic marker expression and alterations in adipogenesis ,11,43,44d people . Our resd people .Low adipogenic potential in ASCs might be involved in metabolic impairments, such as insulin resistance ,42. GiveThe main objective of our study was to evaluate, at first, the impact of SIV/HIV infection per se, on adipose tissue in the absence of ART. In addition to HIV infection, the role of different ART molecules on adipose tissue/adipocytes has been widely evaluated both in vivo and in vitro. First generation thymidine nucleotide reverse transcriptase inhibitors  were associated with the development of clinical lipoatrophy. They were also shown to induce cellular senescence, mitochondrial toxicity and oxidative stress in vitro , and mitThe present study had several limitations. As described above, we found high levels of senescence markers in whole adipose tissue from SIV-infected macaques. Although we showed that ASCs entered senescence in response to HIV proteins in vitro, it is still possible that the senescence observed in adipose tissue in vivo is related to the expression of markers by other adipose tissue cells. Our macaque study has some limitations including the range of duration of chronic infection and the older age of the control group. However, we observed higher features of aging in the adipose tissue of SIV-infected macaques which were younger. We could only show association between Tat and Nef proteins and senescence in vitro. In our in vitro study, we used HIV protein concentration measured in the plasma of naïve HIV-infected subjects which could be higher that the concentration in ART-treated patients. Nevertheless, these are the first results showing that there is a link between the virus itself and aging adipose tissue. Given that our in vitro results indicated that Tat and Nef induced senescence in ASCs, we suggest that there could be a causal link between the virus, HIV proteins, and aging of adipose tissue.Although the senescence in ASCs induced by Nef was associated with impaired adipogenesis, this was not the case for Tat. We hypothesize that Nef’s greater effect on the level of senescence, oxidative stress, and inflammation suggests that there is a threshold for the harmful impact of these changes; however, we did not prove this hypothesis. Furthermore, we did not analyze the impact of NAC on the ASCs’ ability to differentiate into adipocytes. Lastly, it would be interesting to further evaluate the probable relationships between senescence, inflammation, and fibrosis. Since we wanted to study the effect of SIV/HIV on fat in the absence of treatment, we evaluated SIV infection in macaques and the effect of HIV proteins in vitro; however, we did not study adipose tissue obtained from ART-naïve HIV infected patients.SIV/HIV infection induces senescence and associated disorders  in adipose tissue. These changes might be involved in the alterations in fat redistribution and the cardiometabolic diseases observed in aging HIV-infected individuals.


Histoplasma and COVID-19 rapid diagnostics assays were key to the timely diagnosis of both infections, permitting appropriate treatment and patient care.The disease caused by the new SARS-CoV-2, known as Coronavirus disease 2019 (COVID-19), was first identified in China in December 2019 and rapidly spread around the world. Coinfections with fungal pathogens in patients with COVID-19 add challenges to patient care. We conducted a literature review on fungal coinfections in patients with COVID-19. We describe a report of a patient with disseminated histoplasmosis who was likely infected with SARS-CoV-2 and experienced COVID-19 during hospital care in Buenos Aires, Argentina. This patient presented with advanced HIV disease, a well-known factor for disseminated histoplasmosis; on the other hand, we suspected that COVID-19 was acquired during hospitalization but there is not enough evidence to support this hypothesis. Clinical correlation and the use of specific  Coronaviruses are positive-sense RNA viruses and are named for the crown-like spikes on their surface. There are hundreds of coronaviruses, which usually infect animals, birds and various mammals, like camelids, bats, civets, rats, mice, cats and dogs . The firThe first known large outbreak of coronavirus disease occurred in 2002 in the Guangdong province, China. This outbreak of Severe Acute Respiratory Syndrome (SARS) spread to five continents, affecting 8098 people and causing a total of 774 deaths ,6. In DeHistoplasma capsulatum, is frequently reported in the Americas but recently is increasingly reported in regions outside the “traditional” endemic region [Histoplasmosis, a disease caused by the dimorphic thermal fungus c region ,11,12. Hc region .3) and low hemoglobin and hematocrit (10.1 g/dL and 31% respectively) and a normal platelet count . Serum biochemistry analysis was normal. Her HIV viral load was 356,000 copies/mm3 and a CD4 cell count of 3 cells/mm3 . The last ART treatment was in June 2018 . The patient was a heavy smoker, more than 30 cigarettes/day since she was 14 years old and also a person who uses drugs (marijuana and cocaine). Over the two months prior to hospital admission she had developed asthenia and adynamia. On admission (day 1) the patient had fever (temperature 38.2 °C), a respiratory rate of 20 breaths per minute, oxygen saturation of 95%, a heart rate of 106 beats per minute and a blood pressure of 110/80 mm Hg. She presented with hypoventilation and skin and mucosal lesions, including papule-like lesions near her right malar and nose area and an erythematous lesion slightly ulcerated in the central palate. The patient also presented with a bilateral and diffuse micronodular interstitial pattern on the chest computed tomography CT, . Laboratells/mm3 .Histoplasma and Cryptococcus antigen detection  and immunodiffusion method for detection of Histoplasma specific antibodies. Sputum smear microscopy was negative for acid-fast bacilli (AFB) using Ziehl-Neelsen (ZN) stain but intracellular clusters of budding yeast compatible with H. capsulatum were observed using Wright-Giemsa stain  and mycology analysis were collected, including sputum, blood cultures (processed by lysis centrifugation), sera and urine. Specimens were tested for sa stain . Resultsin serum . Based oin serum .Histoplasma antibodies. Renal function parameters showed normal blood levels of creatinine, sodium and potassium and slightly elevated blood urea nitrogen. After 7 days of dAmB therapy, mucosal lesions disappeared and the skin lesions improved and antifungal therapy was switched to itraconazole capsule (200 mg twice daily), as is indicated on the international histoplasmosis guidelines [After three days of dAmB (day 3 of admission), patient became afebrile, with normal heart and respiratory rate and oxygen saturation of 95%. Immunodiffusion testing was non-reactive for idelines . On the Histoplasma antigen testing in urine and serum showed decreasing antigen levels .On day 12, the patient had been afebrile for four days prior, with normal heart and respiratory rate and oxygen saturation of 97%. Blood cultures remained negative. On day 14, the patient continued to be afebrile and follow-up n levels . Further therapy  and skinHistoplasma, aspergillosis, Aspergillus, candidiasis, Candida, Pneumocystis, coccidioidomycosis and Coccidioides and included studies published in English, Spanish and Portuguese. We also did a forward and backward “snowballing” reference chain search. We searched for case reports of cohorts of patients with COVID-19 with co-infection with fungal diseases.A literature search on PubMed Central was done on 31 July 2020. We searched for the following terms—COVID-19; Coronavirus, SARS-CoV-2, fungal infections, mycoses, histoplasmosis, Aspergillus species and one caused by Fusarium proliferatum, three reports described infections caused by yeasts (Candida species and Saccharomyces cerevisiae), two reports of Pneumocystis pneumonia and one report of coccidioidomycosis. Five reports described fungal infections in a hospital series of COVID-19 cases, mostly including infections caused by Aspergillus and Candida species but also cases of mucormycosis and cryptococcosis (A. fumigatus and a few by other Aspergillus species (A. flavus and A. penicillioides). CAPA case series reported prevalence ranging from 8% to 39% and mortality rates ranging from 44% to 67%. All CAPA cases were reported in severe COVID-19 patients  in a patient newly diagnosed with advanced HIV disease and with severe COVID-19. As important findings on this report, the patient presented reticular changes on the chest radiology with alteration of lactate dehydrogenase, a biomarker usually altered with PjP. These findings added the clinical suspicion of PjP and addressing additional laboratory testing for the confirmation of this coinfection [Among other causes of morbidity and mortality in patients with COVID-19, the frequency and impact of fungal co-infections is still limited, although the number of clinical cases and reports are increasing. It is important to note that mortality reported across these studies is concerning. It is still premature to link these fungal co-infections as significant causes of deaths but it should be noted that the risk factors related to mechanical ventilation, prolonged use of corticosteroids and lung damage caused by COVID-19 together with the cytokine storm could set up these patients for invasive fungal diseases ,49,50,51nfection . But, itnfection ,37.Histoplasma antigen testing was crucial in several aspects: (1) Analysis of urine and serum samples represent lower risk of COVID-19 exposure of medical staff to COVID-19; (2) these types of samples are less invasive than other types of samples traditionally used to diagnose histoplasmosis, like BAL or bone marrow aspirate; (3) antigen test was useful for evaluation of the response of the Histoplasma specific antifungal treatment. All these findings correlate with the published guidelines for histoplasmosis diagnosis and treatment in PLHIV [Based on laboratory and epidemiological evidence, we hypothesize that SARS-CoV-2 infection in the patient that we describe in this report happened during hospitalization. But it is important to note that the sensitivity of SARS-CoV-2 RT-PCR in nasopharyngeal swab samples was reported around 70% and it is therefore unknown whether the first negative result was not a false-negative result ,60,61,62in PLHIV ,64.Here we describe a case of disseminated histoplasmosis and COVID-19 in a person with advanced HIV disease. It is well known that advanced HIV is one of the main risks factors for the development of disseminated histoplasmosis. In this patient we suspected that SARS-CoV-2 infection happened during hospitalization but based on information available, it is not possible to proof this hypothesis. Based on this experience, we summarized the main findings of this case and additional evidence published in the literature, aimed to inform other clinicians about fungal co-infection on patients with COVID-19. We highlight the importance of patient’s epidemiological and clinical evaluation, supported by rapid and highly accurate diagnostic assays.


Methods: The PREvention oF thromboembolic events—European Registry in Atrial Fibrillation (PREFER in AF) was a prospective registry including AF patients across seven European countries. We used logistic regression to analyze the relationship between the CHA2DS2-VASc and HAS-BLED scores and outcomes, including mortality, at one year. We evaluated the performance of logistic regression models by discrimination measures (C-index and DeLong test) and calibration measures (Hosmer and Lemeshow goodness-of-fit and integrated discrimination improvement (IDI), with bootstrap techniques for internal validation. Results: In 5209 AF patients with complete information on both scores, average one-year mortality was 3.1%. We found strong gradients between stroke/systemic embolic events (SSE), major bleeding and—specifically—mortality for both CHA2DS2-VASc and HAS-BLED scores, with a similar C-statistic for event prediction. The predictive power of the models with both scores combined, removing overlapping components, was significantly enhanced (p < 0.01) compared to models including either CHA2DS2-VASc or HAS-BLED alone: for mortality, C-statistic: 0.740, compared to 0.707 for CHA2DS2-VASc or 0.646 for HAS-BLED alone. IDI analyses supported the significant improvement for the combined score model compared to separate score models for all outcomes. Conclusions: Both the CHA2DS2-VASc and the HAS-BLED scores predict mortality similarly in patients with AF, and a combination of their components increases prediction significantly. Such combination may be useful for investigational and—possibly—also clinical purposes.Background and Objectives: Atrial fibrillation (AF) is associated with increased mortality, predictors of which are poorly characterized. We investigated the predictive power of the commonly used CHA In a subleeding . Thus, p2DS2-VASc score [2DS2-VASc score of 4, Gallego et al. showed that more deaths (4.5/100 patient-years) occurred for causes other than a thromboembolic (only 1.3 patient-years) or a hemorrhagic event (0.4 patient-years). Here, the HAS-BLED score, besides bleeding, predicted adverse events, including all-cause and cardiovascular mortality [We carried out a comprehensive literature search to investigate published articles regarding the prediction of death through clinical scores derived for AF. We retrieved 25 published articles referring to mortality prediction , WheneveSc score . In seveortality .2DS2-VASc [2DS2-VASc and HAS-BLED. This would have medical relevance due to the current widespread adoption of these scores.Several studies out of AF have shown that scores such as HAS-BLED  and CHA2DS2-VASc ,21,22,23DS2-VASc , have re2DS2-VASc and the HAS-BLED risk scores and outcomes examined . Here, the CHA2DS2-VASc score predicts mortality, in addition to stroke/SSE, better than the HAS-BLED. Our study also confirms the previous literature in showing that statistical predictions are in any case sub-optimal, in the light of the moderately good C-statistic. The risk of stroke/SSE and major bleeding in our study population is slightly higher than in other publications studying the PREFER in AF registry  to the individual components. The usefulness of pursuing a better statistical prediction in spite of reduced practicality—the main advantage of these easy-to-use scores—remains, however, debatable. A novelty here, however, is that the combination of non-duplicated components of the two scores had the highest predictive power for mortality, confirming on the one hand that both ischemic and bleeding events can predict—and in part trigger—the frequent occurrence of death in anticoagulated AF; but also that such components predict triggers of death not easily attributed to thromboembolism or bleeding. The ability to predict such occurrences may allow the focusing on treatment of modifiable risk factors, in addition to only focusing on thrombosis and bleeding, in such populations. Because of the widespread use of the CHA2DS2-VASc and of the HAS-BLED risk score, a combination of their individual components—as shown here—might be useful to risk-stratify patients in epidemiological surveys and prospective trials, as well as, possibly, to better allocate incremental preventive measures.The PREFER in AF registry informed on AF patient profiles in a real-world clinical context, helping to identify specific at-risk patient groups, including those with comorbidities that predispose them to thrombotic events. The current study deriving from that registry is the first that compares and combines a stroke risk score with a bleeding score in order to give the best predictive value on mortality. In line with previously published studies, our report shows strong gradients between the CHA2DS2-VASc and HAS-BLED risk scores, as well as their combination, on mortality, especially compared to the separate risk scores, therefore no re-calibration of coefficients was conducted. We also deliberately did not venture in analyzing causes of death, likely unprecise and not sufficiently controlled in the registry. The relative shortness of the study follow-up is a further limit with a focus on mortality, but data presented here are the first focusing on this endpoint in anticoagulated patients in attempts at improving prediction with a combination of the two most widely used scores. Finally, at the time of the PREFER in AF registry, most patients with AF were treated with VKAs, which does not reflect the current prevalent treatment with NOACs. Whether such limitations affect current applicability of our findings is likely minor, considering the lack of a biologic plausibility (we do not suspect differences in risk prediction once adequate anticoagulation is ensured), and the reassuring results of our sensitivity analyses. The usefulness of the newly proposed combination score should be now, however, further tested in independent cohorts.Although recruitment of consecutive patients at each center was mandatory in PREFER in AF, we cannot exclude biases in patient enrollment and selection, as well as in treatment decisions. Patients excluded from the study sample were fairly numerous; therefore, a limitation is the relatively frequent lack of reporting of risk scores/missing values in the overall registry. Overall, mean age of the study sample was slightly older than in the non-study sample, but the different distribution of other factors  suggests that patient cases included were perhaps more severe than patients excluded. Such considerations limit, in any case, the generalizability of the findings to the unselected PREFER in AF population. Moreover, mortality data could not exactly reflect real mortality, because data were extracted from the comments section of the electronic case-report forms, making death reporting potentially less accurate. However, we estimated that errors in this largely undisputed outcome are unlikely to be numerically relevant. The investigators’ diligence on reporting adverse events was here indeed crucial for the accuracy of data collection. As for model analyses, there were a couple of limitations when evaluating predictive ability using discrimination and calibration measures. Our study was aimed at mainly investigating the predictive power of the CHA2DS2-VASc and the HAS-BLED scores both predict mortality in AF, and a combination of all their components increases prediction significantly. Such combination may be clinically useful. Until now, the main focus of research in AF has been stroke/SSE and bleeding. The availability of robust, real-world data to inform on patients’ risk of death, now numerically more relevant than stroke/SEE in well-anticoagulated patients, will help to better identify strategies to further improve on AF outcomes.Mortality is an important component, so far insufficiently underscored, of the risk connected with AF. The CHA


The Collaborative Cross (CC) mouse population is a valuable resource to study the genetic basis of complex traits, such as obesity. Although the development of obesity is influenced by environmental factors, underlying genetic mechanisms play a crucial role in the response to these factors. The interplay between the genetic background and the gene expression pattern can provide further insight into this response, but we lack robust and easily reproducible workflows to integrate genomic and transcriptomic information in the CC mouse population.We established an automated and reproducible integrative workflow to analyse complex traits in the CC mouse genetic reference panel at the genomic and transcriptomic levels. We implemented the analytical workflow to assess the underlying genetic mechanisms of host susceptibility to diet induced obesity and integrated these results with diet induced changes in the hepatic gene expression of susceptible and resistant mice. Hepatic gene expression differs significantly between obese and non-obese mice, with a significant sex effect, where male and female mice exhibit different responses and coping mechanisms.Integration of the data showed that different genes but similar pathways are involved in the genetic susceptibility and disturbed in diet induced obesity. Genetic mechanisms underlying susceptibility to high-fat diet induced obesity are different in female and male mice. The clear distinction we observed in the systemic response to the high-fat diet challenge and to obesity between male and female mice points to the need for further research into distinct sex-related mechanisms in metabolic disease.The online version contains supplementary material available at 10.1186/s12864-020-07173-x. The Collaborative Cross (CC) is a multiparent genetic reference panel (GRP) of recombinant inbred lines (RIL) of mice derived from eight different founder strains. The CC resource was developed to facilitate the study of the genetic basis of complex traits, and serve as a uniquely powerful resource for the mapping and integration of various phenotypic and genotypic data . CC lineBased on a recent report, people with overweight or obese phenotype account for almost two thirds of the population in the USA . The latHigh-throughput sequencing technologies produce millions of reads in a relatively short time, overcoming the limitations of previous technologies and unravelling previously inaccessible complexities in the transcriptome. However, the overall high complexity of the produced datasets, due to their large size and low signal-to-noise ratio, hinders the interpretation of the underlying information. Most recent analytical methods depend on individual tools that users must download, install and use on their physical drives. The process of deciding which bioinformatic tool accommodates the needs of researchers  can be time-consuming and requires expertise.Our main aim, was to develop an easy-to-use, scalable and cost-effective workflow for the integrative analysis of genotyping and RNA-seq data from CC mice, offering cross-platform portability to different high-end computing configurations. We then opted to use this workflow to assess the underlying QTL that influence genomic susceptibility to high-fat diet induced obesity in mice and the hepatic gene expression response of the mice to high-fat diet and obesity.The tools used in each step of the developed workflow are connected through Python scripts, are archived in our public GitHub repository and can be fully modified. The Python scripts are constructed in a user-friendly manner, so that inexperienced users are required to input only the basic parameters  and the whole process is executed automatically, assuming default parameters. More advanced users can apply different sets of input commands or fully modify the scripts according to their requirements.All bioinformatic tools comprising this workflow have been containerised using Docker containers, resulting in a portable infrastructure that can be executed on physical drives or cloud servers. The main advantage of containerisation is that it is no longer required to install numerous pieces of software, with complex dependencies, downloading instead a pre-built and ready-to-run image file, containing all necessary software and their required dependencies. Another advantage is that applications are run in an isolated and sanitised container environment, where all dependencies are configured for optimal performance, preventing conflicts with other installed programs in the hosting environment. Containerisation ensures the standard operation and performance of applications, not affected by system updates or programming errors from the host end, making the process transparent to the end-user and consistenly fully reproducible. Furthermore, each Docker container can be used as a stand-alone version of the tool making the workflow scalable and adaptable to the individual needs of the researcher.A question that arises is to what extend the start-up, deployment and instantiation of containers performed by the Docker daemon affect the overall performance and the computational cost of a workflow. An IBM research study suggests that the overhead for CPU and memory performance introduced by Docker containers is negligible, and that containerised applications perform equally or better when compared to virtual machine technology . RegardiThe Collaborative Cross mouse lines are novel and highly genetically diverse mouse resource population derived from a genetically diverse set of eight founder mouse strains , designed specifically for complex trait analysis, with the aim to overcome the limitations of previously available resources . A cohor0c (±2). Due to genetic variations between CC lines, breeding rate, the number and sex of litters in each cycle might vary. Therefore, the CC lines were assessed based on litter availability, while making our best efforts to scan as many of the CC lines as possible with representation of both sexes.Mice were weaned at 3 weeks old, housed separately by sex, with a maximum number of five mice per cage, fed with standard rodents’ chow diet  and water ad libitum. All animals were housed at the TAU animal facility at conventional open environment conditions, in clean polycarbonate cages with stainless metal covers, and bedded with wood shavings, at light:dark cycles of 12:12 h, and constant room temperature of 22The study cohort consisted of 540 mice, from 60 different CC lines that were generated, maintained and studied at the TAU small animal facility. Forty-three of lines had representation of both sexes. A subgroup of 84 mice from 43 CC lines, with 20 lines with representation of both sexes, was selected for RNA sequencing.At the age of 8 weeks old, the baseline body weight was recorded. The average body weight (±SE) of females was 18.92 g (±0.27) and of males 22.92 g (±0.24) . ThereafMice were weighed at the beginning of the HFD challenge and bi-weekly thereafter for the following 12 weeks. Mice that gained less than 10 g to their initial body weight over the dietary challenge period were considered “Normal”, while those that gained over 10 g of body weight were considered “Obese”. The median weight at the beginning of the dietary challenge and the median body weight gain for each CC-line are presented in Supplementary Table After overnight recovery from the IPGTT, mice were sacrificed by cervical dislocation, and their livers were collected and stored in liquid nitrogen (− 80 °C). RNA extraction was performed using the QIAGEN commercial kit (Cat.No.73404). Quality control of RNA samples was performed with 2100 BioAnalyzer (Agilent). The RNA Integrity Number (RIN) was used to estimate the integrity of the total RNA sample, samples with RIN above 7.0 passed the quality control test.RNA-seq libraries were prepared using the TruSeq Stranded mRNA library preparation kit (Illumina). Libraries were pooled and sequenced on the Illumina HiSeq 2000 and 2500 sequencers with Illumina v3 sequencing chemistry. Paired-end sequencing was performed by reading 50 bases at each end of a fragment. Overall, each sample consisted of 24 M to 37.5 M RNA-sequencing fragments with an average of 31.5 M fragments.The base images for the Docker containers were pulled from the repositories of Biocontainers and Rocker . For theFor quality assessment of the reads we used the FastQC tool , which iMapping of the reads was performed using the HISAT2 tool, which is a fast and sensitive alignment program for mapping RNA-seq reads . The higFor the purpose of assigning reads to genomic features  we used the software program FeatureCounts (v1.5.0-p1), from the Subread stand-alone package . FeatureP-value adjustment was performed using the Benjamini-Hochberg method and in order to restrict the false discovery rate (FDR) to 0.05, all the genes with adjusted P-values less than 0.05 were selected. The filtering of the gene list  was performed with a custom python script.The DE testing was performed with the R package EdgeR  using a https://bioinfominer.com.The functional pathway analysis was performed with the BioInfoMiner platform . BioInfoAll CC lines were genotyped with high-density SNP markers using the MDA (620 K SNP markers), MUGA (7.7 K markers) and MegaMUGA (77 K markers) genotyping arrays based on the Illumina infinium platform. All SNPs with heterozygous or missing genotypes in the 8 CC founders or not common between the arrays were filtered out, leaving 170,935 SNPs. The SNPs were mapped onto build 37 of the mouse genome. A descent probability distribution was computed using the HAPPY HMM for each of the 170,935 SNPs intervals. The genotype status of the CC lines using the MUGA SNP was presented in CTC 2012 report .The QTL mapping was based on the haplotype mosaic reconstruction with HAPPY. We developed two automated containerised R scripts using the mapping methodology proposed by Durrant and colleagues . The firIt is well documented that obesity and obesity-related health complications are affected by gender, and that sex-specific differences have a genetic basis and cannot be solely attributed to differential hormonal regulation . Gender-In order to assess the presence of confounding factors, we performed a multi-dimensional scaling analysis, using the EdgeR R package. According to the MDS plot Fig.  the poinIn order to partition the samples into non-overlapping groups, we performed a stratified sampling dividing the total sample population into four strata, considering the sex and diabetes status of the mice. Of the total 85 samples, 21 samples were categorised in the Male-Nondiabetic group, 21 samples were categorised in the Male-Diabetic group, 36 samples were categorised in the Female-Nondiabetic group and 5 samples in the Female-Diabetic group. One sample (DT 123) was not used in the analysis due to missing data.We performed haplotype association analysis, using the median value of the weight gain in the 12 weeks of the HFD challenge. We mapped one QTL on chr3 for female mice and one QTL on chr5 for male mice, designated as ObFL and ObML for obesity female locus and obesity male locus, respectively. Details on the position and size of each QTL are given in Table Sgms2 and Hadh that were found to be involved in various processes, such as increased energy expenditure (MP:0004889), decreased susceptibility to diet-induced obesity (MP:0005659) and increased circulating free fatty acid level (MP:0001554). Interestingly, Sgms2 deficiency in mice increases insulin sensitivity and ameliorates high-fat diet-induced obesity and Hadh−/− mice, while having a disrupted β-oxidation pathway, are also protected from diet-induced obesity [Lef1, Dkk2 and Egf, which are all involved in the Wnt signaling pathway (GO:0016055). Non-canonical Wnt signaling has been shown to contribute to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation [In female mice, prioritised genes included  obesity , 30. Othammation .Ppargc1a, which is found to be involved in various enriched processes both in GO and in MGI Mammalian Phenotype Ontology, including decreased muscle weight (MP:0004232), regulation of muscle tissue development (GO:1901863) and lipid modification (GO:0030258). Ppargc1a is a transcriptional co-activator that regulates genes involved in energy metabolism through its interaction with Pparγ. In human GWAS analysis a single nucleotide variant of Ppargc1a (rs8192678) has been associated with susceptibility to obesity and insulin resistance [Cckar, found involved in processes such as abnormal small intestinal transit time (MP:0006002) and abnormal intestinal cholesterol absorption (MP:0002645). Rats with a naturally occurring mutation in Cckar (Otsuka Long-Evans Tokushima Fatty (OLETF) rat) develop diabetes and obesity [Sod3 is involved along with Ppargc1a in: response to reactive oxygen species (GO:0000302), increased susceptibility to injury (MP:0005165), and abnormal cytokine secretion (MP:0003009). Over-expression of Sod3 in high-fat diet fed mice has been shown to block diet induced obesity [Med28 is involved in the regulation of muscle cell differentiation (GO:0051147). Med28 is one of the subunits of the Mediator complex, which acts as a transcription factor co-activator and plays an important role in muscle metabolism by enhancing the transcriptional activity of Ppargc1a and Pparα [Slit2 is also prioritised and shares enriched terms with Ppargc1a, such as regulation of smooth muscle cell migration (GO:0014910) and response to organonitrogen compound (GO:0010243). Slit2 has been shown to regulate metabolic function and thermogenic activity and improve glucose homeostasis in diet-induced obese mice, mainly through Ucp1 [In male mice the most highly prioritised gene is  MP:00042, regulat obesity . Priorit obesity . Med28 ind Pparα . Lastly,ugh Ucp1 , which hDE analysis was performed on the Male-Nondiabetic and Female-Nondiabetic groups separately. In order to perform the statistical differential expression tests we divided the samples into two subgroups, 10 samples were categorised in the Male-Nondiabetic-Obese group , 11 samples were categorised in the Male-Nondiabetic-Normal  12 samples were categorised in the Female-Nondiabetic-Obese group  and 24 samples in the Female-Nondiabetic-Normal group . Differences in library size were addressed by TMM normalisation.Lepr and Ppargc1a that were under-expressed in obese females and Pnlip, Pyy, and Ins2 that were over-expressed in obese females. Lepr functions as a receptor for leptin, an adipose secreted hormone that regulates energy expenditure, satiety, lipid and glucose metabolism and immune system activation. Pancreatic lipase is normally secreted from the pancreas and is efficient in the digestion of dietary fats. Inhibition of Pnlip may prevent HFD induced obesity in mice. Orlistat, an inhibitor of Pnlip was the first FDA-approved anti-obesity therapeutic drug in treating diet-induced obesity [Pnlip has been observed in mice induced by fasting via the PPARα-FGF21 signalling pathway [Ins2 ectopic expression in the liver has been observed before in mice subjected to HFD [Pyy may be expressed ectopically in the liver in the same way as Ins2 in response to the HFD.As a first step we tried to assess the DE genes within the two genders, performing the DE tests between control and case groups of the same gender. The Female-Nondiabetic-Normal samples (control group) versus the Female-Nondiabetic-Obese samples (case group) DE genes list consisted of 1382 DE genes. Using BioInfoMiner for the enrichment analysis we obtained two prioritised gene list of 23 DE genes from GO and 21 DE genes from MGI Mammalian Phenotype. Seven genes were common in both gene lists. Processes that were highly enriched in DE genes in female mice include digestion (GO:0007586), regulation of insulin secretion (GO:0050796), response to lipid GO:0033993), fatty acid biosynthetic process (GO:0006633), increased energy expenditure (MP:0004889), decreased susceptibility to diet-induced obesity (MP:0005659), abnormal glucose tolerance (MP:0005291) and decreased circulating leptin level (MP:0005668). Top prioritised, linker genes in female mice include  obesity . Increas93, fattyd to HFD . We hypoNos1, Ryr1, Des, Ttn all under-expressed in obese males while Ryr2 is over-expressed. Ryr1 is mainly expressed in skeletal muscle. The encoded protein functions as a calcium release channel in the sarcoplasmic reticulum. However, there is a number of studies suggesting that RyRs are widely expressed and have been linked with inositol 1,4,5-trisphosphate receptors in hepatocytes [Ryr1 could lead to reduced release of Ca2+ from the sarcoplasmic (muscle cells) and the endoplasmic reticulum (hepatic cells) into the cytoplasm and therefore hinder muscle contraction as well as the regulation of mitochondrial metabolism and glycogen degradation [Des encodes a muscle-specific class III intermediate filament, which is important to help maintain the structure of sarcomeres and has been linked with hepatic fibrosis due to obesity [Titin is an essential component of skeletal and cardiac muscles, but it has been suggested in recent studies that titin isoforms are expressed in non-muscle tissues including the liver, with an essential role in maintaining cellular organisation and contributing to signal transduction [Ryr2 is primarily expressed in cardiac muscle. Ryr2 channels are associated with mitochondrial metabolism, gene expression regulation and cell survival, in addition to their role in cardiomyocyte contraction. A recent study links Ryr2 to insulin release and glucose homeostasis, suggesting that the upregulation of Ryr2 might be a coping mechanism in response to stress [Nos1 in obesity and diabetes is largely attributed to insulin resistance [The DE genes list for the Male-Nondiabetic-Normal samples (control group) versus the Male-Nondiabetic-Obese samples (case group) consisted of 1589 DE genes. We performed functional analysis on this list and obtained two prioritised gene lists of 24 DE genes from GO and 22 genes from MGI Mammalian Phenotype. Ten of the genes from the two prioritised gene lists were common in both. Enriched pathways in male mice are overwhelmingly related to muscle and cardiac muscle processes: impaired muscle contractility (MP:0000738), abnormal skeletal muscle mass (MP:0004817), myopathy (MP:0000751), cardiac muscle hypertrophy (GO:0003300). A second category of enriched processes involves ion transport (GO:0006811). Top prioritised linker genes in male mice include atocytes . Downregradation , 43. Des obesity . Titin isduction . Ryr2 iso stress . Nos1 prsistance .Finally, in order to assess the DE genes between the two genders we performed DE tests between groups that belonged to different genders. The DE genes list for the Male-Nondiabetic-Normal samples versus the Female-Nondiabetic-Normal samples comparison, consisted of 1923 genes. Functional analysis produced two prioritised gene lists consisting of 28 genes from GO and 25 genes from MGI Mammalian Phenotype, with 9 common genes. As for the DE genes for the Male-Nondiabetic-Obese samples versus the Female-Nondiabetic-Obese samples, the genes list consisted of 1940 DE genes. Functional analysis produced two prioritised gene lists consisting of 40 genes from GO and 22 genes from MGI, with 8 common genes. Between the two groups 482 DE genes are shared while 1441 genes are uniquely DE in the Male-Nondiabetic-Normal versus the Female-Nondiabetic-Normal samples and 1458 genes are uniquely DE in the Male-Nondiabetic-Obese versus the Female-Nondiabetic-Obese samples.Cav1, Egfr, Gstp1, Gcg, and Nox4 are consistently over-expressed in male versus female mice both in the non-obese and obese comparisons. Caveolin-1 regulates hepatic lipid accumulation and glucose metabolism and plays an important role in metabolic adaptation [Esr1, Il1b and Ptgs2 are consistently over-expressed in female versus male mice in both comparisons. Ptgs2 is involved in inflammation in fat and drives obesity-linked insulin resistance and fatty liver [Tph1 is under-expressed in non-obese males and over-expressed in obese males in comparison to females. In contrast, Ryr2 is over-expressed in non-obese males and under-expressed in obese males in comparison to females. Tph1 is involved in the synthesis of serotonin, which is known to modulate appetite, energy expenditure and thermogenesis. Tph1-deficient mice fed a high-fat diet are protected from obesity, insulin resistance and NAFLD [The 482 DE genes that are common in the two comparisons are enriched in genes that are involved in processes such as the epoxygenase P450 pathway (GO:0019373), long-chain fatty acid metabolic process (GO:0001676), negative regulation of gluconeogenesis (GO:0045721), increased circulating gonadotropin level (MP:0003362), and regulation of NF-κB import into nucleus (GO:0042345). Functional analysis of these 482 DE expressed genes produced 21 prioritised genes from GO and 20 prioritised genes from MGI Mammalian Phenotype. Of these 8 genes were common in both lists. aptation . Esr1, Ind NAFLD .Il2, Il4, Il10, and Il1r1, were predominantly over-expressed in female mice. Vdr and Lepr were also over-expressed in females. Males had over-expressed Mrap2 and Oprm1. Mrap2 encodes a protein that modulates melanocortin receptor signalling. Mice deficient in Mrap2 exhibit severe obesity and a mutation in this gene may be associated with severe obesity in human patients [Functional analysis of the 1441 genes that were uniquely DE in the Male-Nondiabetic-Normal versus the Female-Nondiabetic-Normal comparison produced enriched processes like abnormal bone volume (MP:0010874), increased mesenteric fat pad weight (MP:0009298), unsaturated fatty acid and lipid metabolic process , regulation of hormone levels (GO:0010817), and a number of terms related to inflammatory processes, such as abnormal IgG2a level (MP:0020176), increased susceptibility to autoimmune diabetes (MP:0004803) and small intestinal inflammation (MP:0003306). The list of prioritised genes consisted of 21 genes from GO and 23 genes from MGI, with 9 overlapping genes. Genes involved in inflammatory processes, such as patients .Ifng, Il6, and Tnf. Other enriched processes were muscle system processes (GO:0003012) and abnormal muscle contractility (MP:0005620), abnormal adrenaline level (MP:0003962), abnormal blood pH regulation (MP:0003027), digestion (GO:0007586) and decreased glucagon secretion (MP:0002711). Genes involved in these processes were predominantly under-expressed in male mice, for example, the Mc2r and Pyy. Notably, both Ins1 and Ins2 are also highly over-expressed in obese female mice compared to an almost zero level of expression in male mice. Mc2r belongs to the MCR family that plays an important role in appetite and energy regulation via leptin signalling. Increased MCRs expression in the liver has been associated with muscle tissue damage and potentially exerts a protective effect through metabolic regulation [Finally, functional analysis of the 1458 unique DE genes from the Male-Nondiabetic-Obese versus the Female-Nondiabetic-Obese comparison resulted again in enriched immune response related processes (GO:0006955) and positive regulation of inflammatory response GO:0050729) with related genes also predominantly over-expressed in obese females. The list of prioritised genes consisted of 34 genes from GO and 20 genes from MGI Mammalian Phenotype, with 4 overlapping genes. Genes related to immunological processes include gulation . The com9 with reObesity encompasses a complex array of traits closely related to the development of type 2 diabetes, metabolic syndrome and associated with comorbidities such as cardiovascular disease, hypertension, atherosclerosis and various cancers. In this study we have used a systems biology approach to examine genetic susceptibility and hepatic gene response to obesity in CC mice. The CC panel is a unique resource with wide genetic diversity that enables complex trait genomic mapping at a very high resolution. Moreover, it provides the opportunity to study differences in genetic expression in a diverse genetic reference population under controlled environmental conditions.To facilitate this analysis, we developed a unified, automated workflow using Docker containers that can be simultaneously deployed on different computation infrastructures and can facilitate cross-platform collaboration. This is achieved by creating an identical insulated and stable operating environment that can be precisely controlled resulting in consistency overtime, without the worry of conflicting dependencies, UNIX compatibility issues and system updates. Docker containers can be used effectively to address some of the major setbacks of microarray and next generation sequencing technologies. The fast start-up time of Docker containers opens up the ability to chain together the execution of multiple containers and the development of sophisticated workflows for genomic analyses.This work takes into consideration the overall analytical performance of the workflow as well as computational cost. Our aim was to develop an automated, accurate, and computationally efficient analytical process, while keeping the computational requirements at a minimum. Exploiting the minimal performance loss introduced by the Docker engine we developed a modular architecture for the integrative analysis of QTL and RNA-seq data that can be run on a personal computer (with approximately 4 GB of RAM or higher). For the aforementioned purpose the bioinformatic tools have been picked according to their overall effectiveness and their computational cost as described in the methods. The workflow we propose requires no more than 4.3 GB of RAM, 10 GB of physical drive and can run a top-down analysis of 20 GB of input data in less than 3 h .Ppargc1a was suggested to be a candidate gene through comparative genomics and haplotype analysis [Ppargc1a expression is estrogen regulated and has a protective effect against obesity-induced oxidative damage [Ppargc1a is under-expressed in obese versus normal females. Additional genes involved in pathways closely related to the obesity phenotype highlighted by our analysis as potential candidates that play a role in male mice susceptibility to obesity are Cckar, Sod3, Med28, and Slit2.Using our workflow, we mapped two QTLs related to the body weight gain phenotype, one on chromosome 5 for male mice and one on chromosome 3 for female mice. The QTL on chromosome 5 has already been described as significant in relation to the percentage of body fat, in response to an atherogenic diet, where analysis . There he damage , 56. In Cyp2u1, a hydroxylase that is involved in the metabolism of long chain fatty acids. However, the two gene lists derived from the QTL and differential expression analysis share a high number of common relevant enriched pathways, such as increased energy expenditure (MP:0004889), decreased susceptibility to diet-induced obesity (MP:0005659) and increased circulating free fatty acid level (MP:0001554).QTLs on chromosome 3 have been previously described to be involved in the percentage of body fat and heat loss, without the introduction of a high fat diet , 58. To The comparison of male and female DE results  at the start of the dietary challenge, body weight gain after 12 weeks (ΔBW0-12) of dietary challenge, aNA blood glucose levels at T180 after IPGTT challeng (BGM-T180) for all mice included in the analysis.Additional file 3:Table S3. List of differentially expressed genes from the female non-diabetic normal vs. female non-diabetic obese mice with the logFC and adjusted p-values.Additional file 4:Table S4. List of differentially expressed genes from the male non-diabetic normal vs. male non-diabetic obese mice with the logFC and adjusted p-values.Additional file 5:Table S5. List of differentially expressed genes from the male non-diabetic normal vs. female non-diabetic normal mice with the logFC and adjusted p-values.Additional file 6:Table S6. List of differentially expressed genes from the male non-diabetic obese vs. female non-diabetic obese mice with the logFC and adjusted p-values.


Hypertension and/or myocardial infarction are common causes of heart failure in Type 2 diabetes. Progression to heart failure is usually preceded by ventricular dysfunction, linked to matrix metalloproteinase (MMP) mediated extracellular matrix changes. We hypothesise that the minor allele of genetic variant rs3918242 in the promoter region of the MMP-9 gene is associated with hypertension and/or myocardial infarction, with resultant progression of dysfunctional cardiac remodelling in patients with diabetes without symptomatic heart failure.We genotyped 498 diabetes patients participating in the St Vincent’s Screening TO Prevent Heart Failure (STOP-HF) follow-up programme for the rs3918242 single nucleotide polymorphism and investigated associations with the co-primary endpoints hypertension and/or myocardial infarction using a dominant model. We also evaluated resulting cardiometabolic phenotype and progression of ventricular dysfunction and cardiac structural abnormalities over a median follow-up period of 3.5 years.p = 0.026] over a median follow-up of 3.5 years . Conversely, rs3918242 was not associated with hypertension, blood pressure, pulse pressure or left ventricular mass index at baseline or over follow up.The CT/TT genotype comprised 28.1% of the cohort and was associated with a twofold higher risk of myocardial infarction (17.9% vs 8.4%), a reduction in ejection fraction and greater left ventricular systolic dysfunction progression [adjusted OR = 2.56 , Diabetes patients with the minor T allele of rs3918242 in the STOP-HF follow up programme have greater risk of myocardial infarction, lower ejection fraction and greater progression of left ventricular systolic abnormalities, a precursor to heart failure. These data may support further work on MMP-9 as a biomarker of ventricular dysfunction and the investigation of MMP-9 inhibitors for heart failure prevention in diabetes, particularly in the post-infarction setting.ClinicalTrials.gov Identifier: NCT00921960 The growth of cardiovascular disease in diabetes, including hypertension, myocardial infarction and diabetes-related cardiomyopathy, is a persistent global public health problem , 2. In phttps://www.ncbi.nlm.nih.gov/snp/?term=rs3918242) near the promoter region of the MMP-9 gene has been associated with increased circulating levels of MMP-9 and with higher frequencies of vascular disease, especially in type 2 diabetes . There were no differences between the two groups in terms of LVDD, LVD or LVD and structural cardiac abnormality progression. There was also a significant difference between both groups in the proportion of patients demonstrating progression of LAVI , although this did not remain significant when adjusted for age and sex.Furthermore, over a median follow-up of 3.5 years  there was a significant difference in LVSD progression ; death or MACE or LVD progression ; death or MACE or LVD or structural cardiac abnormality progression .Although myocardial infarction occurred in only 11% of the total population, the majority of patients with LVSD at baseline (54%) or with LVSD progression (58%) had a history of MI. Due to the strong association between the minor T allele, myocardial infarction and reduced LVEF in our study, we carried out a post-hoc analysis of patients with a history of myocardial infarction to see if severity of cardiac damage, denoted by reduced LVEF, was greater in this subset. We compared clinical characteristics, medications and Doppler echocardiography of the patients with myocardial infarction according to CC or CT/TT genotype  allele of rs3918242 we observed a twofold risk of prior myocardial infarction, but no associated risk of hypertension. We also found significantly lower ejection fraction at baseline in CT/TT genotyped patients and greater progression of left ventricular systolic dysfunction over a median 3.5 year follow up period, suggesting higher risk of the future development of HF. Consistent with these observations, in propensity-matched, sub-group analyses of patients with a history of myocardial infarction, CT/TT genotype patients had significantly reduced ejection fraction compared to CC controls, with greater reduction in ejection fraction compared to T allele patients in the overall cohort. As MMP-9 is an inducible gelatinase and rs3918242 C > T in the promoter region of the gene is known to augment expression of MMP-9, these data may support the use of MMP-9 as a biomarker of ventricular dysfunction and the investigation of MMP-9 inhibitors for HF prevention in diabetes, particularly in the post-infarction setting.–8 and consequently identify only a minority of gene variants associated with disease risk. Moreover, GWAS may lack deep phenotypic characterisation and many of the variant candidates emerging from GWAS to date have uncertain functional significance [The observational nature of this study means that causality between rs3918242, myocardial infarction and ejection fraction cannot be definitively established. In addition, the population is exclusively Irish Caucasian, and our results may not hold in populations with different ethnicities, where minor allele frequencies differ. The work is also limited by relatively low patient number and potentially biased by the absence of a matched non-diabetic cohort to investigate whether this relationship applies only in the context of diabetes. Furthermore, while the minor T allele of rs3918242 is associated with coronary artery and cerebrovascular diseases in large clinical studies and a recent meta-analysis, , 27, 28 ificance . A pragmificance , 30.The main strength of this study is the detailed clinical, biochemical and deep-phenotyping with sequential Doppler echocardiography to highlight the association of myocardial infarction, asymptomatic left ventricular dysfunction and progression of left ventricular systolic dysfunction amongst carriers of the rs3918242 T allele. The data may be important in the context of concerns about the burden of heart failure in diabetes and support the potential of MMP-9 as a diagnostic marker and/or therapeutic target post-MI in order to prevent heart failure in this setting. Our study is also consistent with a large population based study of coronary artery disease patients, which showed that the rs3918242 T-allele associated with the risk of clinical events only in patients with metabolic syndrome . ChronicIn contrast, we did not find any association of rs3918242 with baseline prevalence of hypertension, blood pressure and pulse pressure. This is unlike previous studies showing MMP-9 is associated with arterial stiffness in patients with diabetes . In an eAlternatively, the present study may support a hypothesis that over-expression of MMP-9 is most associated with the evolution of, and cardiac response to, myocardial infarction in diabetes. The magnitude of EF difference at baseline, while significant, was considerably smaller in the total population rather than the prior MI subset and the majority of patients that demonstrated LVSD progression over time had prior MI. This relationship may be complex, involve the innate immune response and have important temporal features. For example, in accordance with our study, maximal plasma MMP-9, seen early in the first 12 h post-MI, is independently related to lower EF and increased circulating leukocytes . On one Future avenues of research could build on these data with a view to individualizing care. For example, excess MMP-9 activity associated with the minor allele of rs3918242 may be a therapeutic target in post-MI diabetes patients. To date, MMP inhibitors have produced mixed results when evaluated clinically in cardiovascular disease patients including post-MI, which may relate to temporal issues described above, as well as a lack of selectivity of inhibitors for pathophysiological MMP isoforms . HoweverDiabetes patients with the minor T allele of rs3918242 in the STOP-HF follow up programme have greater prevalence of myocardial infarction, reduced ejection fraction at baseline and greater progression of left ventricular systolic dysfunction over a median 3.5-year follow-up period. The reduced ejection fraction is lower in subsets of post-myocardial infarction patients with the CT/TT genotype. In contrast, we did not find any association of rs3918242 with baseline prevalence of hypertension, blood pressure and pulse pressure. These data may support further work on MMP-9 as a biomarker of ventricular dysfunction and the investigation of MMP-9 inhibitors for HF prevention in diabetes, particularly in the post-infarction setting.Additional file 1. Supplementary Data File.