MEK/ERK activation plays a decisive role in Zika virus morphogenesis and release.

Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication.

Evaluation of kinase inhibitors as potential therapeutics for flavivirus infections.

The recent introduction of Zika virus (ZIKV), the recurrence of dengue virus (DENV), and the lethality of yellow fever virus (YFV) have had a significant impact on Brazilian society and public health. Here, we targeted two cellular kinases implicated in cell proliferation and cancer that are also important for viral replication: mitogen-activated protein kinase kinase (MEK) and Src. We used two MEK inhibitors - trametinib and selumetinib - and two Src inhibitors - saracatinib and bosutinib - to inhibit ZIKV, DENV, and YFV replication in cell culture. The cytotoxicity of the four inhibitors was determined by the observation of abnormal morphology and quantification of adherent cells by crystal violet staining. The antiviral activity of these drugs was assessed based on the reduction of plaque-forming units in cell culture as evidence of the inhibition of the replication of the selected flaviviruses. All four inhibitors showed antiviral activity, but among them, trametinib was the safest and most efficacious against all of the viruses, inhibiting the replication of ZIKV and YFV by 1000-fold, and DENV2/3 by nearly 100-fold. This pan-antiviral effect shows that trametinib could be repurposed for the treatment of flaviviral infections.

A study of the MAYV replication cycle: Correlation between the kinetics of viral multiplication and viral morphogenesis.

Mayaro virus (MAYV) is mainly found in Central and South America and causes a febrile illness followed by debilitating arthritis and arthralgia similar to chikungunya virus (CHIKV). Infection leads to long-term sequelae with a direct impact on the patient's productive capacity, resulting in economic losses. Mayaro fever is a neglected disease due to the limited epidemiological data. In Brazil, it is considered a potential public health risk with the number of cases increasing every year. Most of our knowledge about MAYV biology is inferred from data obtained from other alphaviruses as well as more recent studies on MAYV. Here, we analyzed the kinetics of viral replication through standard growth curves, quantification of intracellular and extracellular particles, and RNA quantification. We compared transmission electron microscopy data during different stages of infection. This approach allowed us to establish a chronological order of events during MAYV replication and its respective timepoints including cell entry through clathrin-mediated endocytosis occurring at 15-30 min, genome replication at 2-3 h, morphogenesis at 4 hpi, and release at 4-6 hpi. We also present evidence of uncharacterized events such as ribosome reorganization as well as clusters of early viral precursors and release through exocytosis in giant forms. Our work sheds new and specific light on the MAYV replication cycle and may contribute to future studies on the field.

A20-binding inhibitor of nuclear factor-kappaB (NF-kappaB)-2 (ABIN-2) is an activator of inhibitor of NF-kappaB (IkappaB) kinase alpha (IKKalpha)-mediated NF-kappaB transcriptional activity.

NF-κB transcription factors are pivotal players in controlling inflammatory and immune responses, as well as cell proliferation and apoptosis. Aberrant regulation of NF-κB and the signaling pathways that regulate its activity have been involved in various pathologies, particularly cancers, as well as inflammatory and autoimmune diseases. NF-κB activation is tightly regulated by the IκB kinase (IKK) complex, which is composed of two catalytic subunits IKKα and IKKß, and a regulatory subunit IKKγ/NEMO. Although IKKα and IKKß share structural similarities, IKKα has been shown to have distinct biological functions. However, the molecular mechanisms that modulate IKKα activity have not yet been fully elucidated. To understand better the regulation of IKKα activity, we purified IKKα-associated proteins and identified ABIN-2. Here, we demonstrate that IKKα and IKKß both interact with ABIN-2 and impair its constitutive degradation by the proteasome. Nonetheless, ABIN-2 enhances IKKα- but not IKKß-mediated NF-κB activation by specifically inducing IKKα autophosphorylation and kinase activity. Furthermore, we found that ABIN-2 serine 146 is critical for the ABIN-2-dependent IKKα transcriptional up-regulation of specific NF-κB target genes. These results imply that ABIN-2 acts as a positive regulator of NF-κB-dependent transcription by activating IKKα.

Epidemiological Analysis of COVID-19 Cases in Native Amazonian Communities from Peru.

Despite early control measures, SARS-CoV-2 reached all regions of Peru during the first wave of the pandemic, including native communities of the Peruvian Amazon. Here, we aimed to describe the epidemiological situation of COVID-19 in the Amazonas region of Peru using an open database of 11,124 COVID-19 cases reported from 19 March to 29 July 2020, including 3278 cases from native communities. A high-incidence area in northern Amazonas (Condorcanqui) reported a cumulative incidence of 63.84/1000 inhabitants with a much lower death rate (0.95%) than the national average. Our results showed at least eight significant factors for mortality, and the Native Amazonian ethnicity as a protective factor. Molecular confirmatory tests are necessary to better explain the high incidence of antibody response reported in these communities.

A phase 1 trial of the histone deacetylase inhibitor AR-42 in patients with neurofibromatosis type 2-associated tumors and advanced solid malignancies.

PURPOSE: Given clinical activity of AR-42, an oral histone deacetylase inhibitor, in hematologic malignancies and preclinical activity in solid tumors, this phase 1 trial investigated the safety and tolerability of AR-42 in patients with advanced solid tumors, including neurofibromatosis type 2-associated meningiomas and schwannomas (NF2). The primary objective was to define the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs). Secondary objectives included determining pharmacokinetics and clinical activity. METHODS: This phase I trial was an open-label, single-center, dose-escalation study of single-agent AR-42 in primary central nervous system and advanced solid tumors. The study followed a 3 + 3 design with an expansion cohort at the MTD. RESULTS: Seventeen patients were enrolled with NF2 (n = 5), urothelial carcinoma (n = 3), breast cancer (n = 2), non-NF2-related meningioma (n = 2), carcinoma of unknown primary (n = 2), small cell lung cancer (n = 1), Sertoli cell carcinoma (n = 1), and uveal melanoma (n = 1). The recommended phase II dose is 60 mg three times weekly, for 3 weeks of a 28-day cycle. DLTs included grade 3 thrombocytopenia and grade 4 psychosis. The most common treatment-related adverse events were cytopenias, fatigue, and nausea. The best response was stable disease in 53% of patients (95% CI 26.6-78.7). Median progression-free survival (PFS) was 3.6 months (95% CI 1.2-9.1). Among evaluable patients with NF2 or meningioma (n = 5), median PFS was 9.1 months (95% CI 1.9-not reached). CONCLUSION: Single-agent AR-42 is safe and well tolerated. Further studies may consider AR-42 in a larger cohort of patients with NF2 or in combination with other agents in advanced solid tumors. TRIAL REGISTRATION: NCT01129193, registered 5/24/2010.

Invasive pulmonary aspergillosis in a patient with cirrhosis.

Aspergillus molds are ubiquitous environmental molds that can cause devastating invasive infections in immunocompromised patients. These infections often go unrecognized in critically ill patients. This case describes a 68 year-old female resident of a long-term nursing facility with history of dementia, nonalcoholic fatty liver disease with cirrhosis, chronic kidney disease stage III and insulin-dependent type 2 diabetes who presented with vomiting, diarrhea and leg swelling. She developed hypotension and was treated for sepsis but found to have negative routine infectious workup. Chest imaging showed nodular densities and bilateral opacities. She developed acute renal failure and hypoxic respiratory failure followed by acute decompensated cirrhosis with refractory volume overload and hypotension and was eventually transitioned to comfort care measures. Autopsy ultimately showed invasive pulmonary aspergillosis. Here we review the diagnosis and management of invasive fungal infections in critically ill patients without typical risk factors or clinical findings for invasive fungal disease. Invasive fungal infections are frequently missed and carry high mortality rates, therefore warranting consideration in critically ill populations.

Seroprevalence and risk factors of bovine leptospirosis in the province of Manabí, Ecuador.

Leptospirosis is a zoonotic disease with worldwide distribution. The disease affects dairy and beef cattle, causing infertility, abortion, and reduced milk yield. A cross-sectional study was conducted to determine the seroprevalence of leptospirosis in cattle and the associated risk factors in the province of Manabí, Ecuador. Serum samples from 749 animals from 55 cattle herds were analyzed using the microscopic agglutination test (MAT). Animals were considered positive when titers were ≥ 1:100. The association between the potential risk factors and the positive Leptospira result was modeled at both animal and herd level using a generalized linear model with a binomial distribution and logarithmic link. The seroprevalence was 56.21% at the individual level and 98.18% at the herd level. The most prevalent serovars were Pomona (28.57%) and Icterohaemorragiae (22.30%). At the animal level, only the age was associated with leptospirosis seropositivity. Seroprevalence in animals over three years of age was 1.197 (95% confidence intervals (CI), 1.032 - 1.390), higher compared to animals up to three years old. The seroprevalence of Leptospira spp. was higher in farms with no veterinary assistance (PR = 1.209; 95%CI 1.053 - 1.388) and without a vaccination program against Leptospira (PR = 1.399; 95%CI 1.09 - 1.794). In addition, herds from Junín canton had a significantly higher seroprevalence of Leptospira spp (PR = 1.548; 95%CI 1.213 - 1.977) compared to the Bolívar canton, which had the lowest seroprevalence. In conclusion, more than half of the animals were positive to Leptospiraspp, and almost all herds had at least one positive animal. Furthermore, veterinary assistance and vaccination of cattle must be considered as essential aspects of the disease control program.

Demographic and clinical characteristics of chikungunya patients from six Colombian cities, 2014-2015.

In 2014, the chikungunya virus reached Colombia for the first time, resulting in a nationwide epidemic. The objective of this study was to describe the demographics and clinical characteristics of suspected chikungunya cases. Chikungunya infection was confirmed by enzyme-linked immunosorbent assay and 548 patients where included in the study. Of these patients, 295 were positive for antibodies against chikungunya (53.8%), and 27.6% (151/295) were symptomatic for chikungunya infection, with a symptomatic:asymptomatic ratio of 1.04:1. Factors associated with infection included low income and low socio-economic strata (odds ratio [OR]: 1.8; 95% confidence interval [CI]: 1.0-3.2, p = 0.003 and OR: 2.1; CI: 1.3-3.4, p = 0.002, respectively). Confirmed symptomatic cases were associated with symmetric arthritis (OR: 11.7; CI: 6.0-23.0, p < 0.001) of ankles (OR: 8.5; CI: 3.5-20.9, p < 0.001), hands (OR: 8.5; CI: 3.5-20.9, p < 0.001), feet (OR: 6.5; CI: 2.8-15.3, p < 0.001), and wrists (OR: 17.3; CI: 2.3-130.5, p < 0.001). Our study showed that poverty is associated with chikungunya infection. Public health strategies to prevent and control chikungunya should focus on poorer communities that are more vulnerable to infection. The rate of asymptomatic infections among confirmed cases was 48.8%. However, those with symptoms displayed a characteristic rheumatic clinical picture, which could help differentiate chikungunya infection from other endemic viral diseases.

Analysis of the risk of infection in patients with chronic lymphocytic leukemia in the era of novel therapies.

We studied the risk of infections in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL). Major infections were defined as requiring hospital admission or intravenous antimicrobial treatment. Incidence rate (IR) ratios (IRR) were used to compare infection rates. Of 263 CLL patients followed for 936.9 person-years, 60% required treatment for progressive CLL (66 received ibrutinib). Infections occurred in 71.9% patients (IR 92.4/100 person-years) with 31.9% having major infections (IR 20.3/100 person-years) and infections causing 37.5% of deaths. CLL treatment was associated with significantly higher risk of major (IRR 3.31, 95% CI 2.10, 5.21) and minor (IRR 1.78, 95% CI 1.43, 2.22) infections. Compared to their previous chemoimmunotherapy patients receiving salvage ibrutinib therapy (n = 47) had a significantly increased risk of a major infection (IRR 2.35 95% CI 1.27, 4.34). The risk of infection in CLL patients remains high even with use of less immunosuppressive therapies.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.

Cardiac rehabilitation participation in underserved populations. Minorities, low socioeconomic, and rural residents.

Cardiac rehabilitation (CR) services in the United States are underutilized and participation is particularly low for racial and ethnic minorities, low socioeconomic status patients, and rural residents. Reduced participation may not only indicate a failure in transitional cardiac care during the in hospital referral process but also could be due to barriers attributed to patients, providers, employers, or medical systems. In-depth analysis of this problem is impeded by difficulties with the identification of underserved groups in clinical settings. Disparities in CR participation certainly contribute to poor medical outcomes in these populations that stand to benefit greatly from lifestyle modifications. It is critical that CR providers survey their communities for underserved populations and coordinate creative efforts aimed at overcoming barriers to participation. Moreover, it is likely that referral to, and participation in, CR will soon be considered a quality indicator, providing further incentive for programs to optimize CR utilization among all eligible patients.