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STIM1 is a Ca2+ sensor within the ER membrane known to activate the plasma membrane store-operated Ca2+ channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca2+ signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca2+ sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies. [ABSTRACT FROM AUTHOR]

Background: Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. Methods/Principal Findings: We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. Conclusions/Significance: Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak. [ABSTRACT FROM AUTHOR]

Background: Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available. Methodology/Principal Findings: Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274-281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitopeblocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization. Conclusions/Significance: The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera. [ABSTRACT FROM AUTHOR]

BACKGROUND:Guidelines recommend integrating hypertension screening for HIV-infected adults, but blood pressure measurements may be dynamic around the time of HIV testing. METHODS:We measured a seated resting blood pressure in adults (≥18 years) prior to HIV testing, and again after receiving HIV test results, in an ambulatory HIV clinic in KwaZulu-Natal, South Africa. We assessed sociodemographics, smoking, body mass index, diabetes, substance abuse, and anxiety/depression. We used blood pressure categories defined by the Seventh Joint National Committee (JNC 7) classifications, which includes normal, pre-hypertension, stage 1 hypertension, and stage 2 hypertension. RESULTS:Among 5,428 adults, mean age was 31 years, 51% were male, and 35% tested HIV-positive. Before HIV testing, 47% (2,634) had a normal blood pressure, 40% (2,225) had prehypertension, and 10% (569) had stage 1 or 2 hypertension. HIV-infected adults had significantly lower blood pressure measurements and less hypertension, as compared to HIV-negative adults before HIV testing; while also having significantly elevated blood pressures after HIV testing. In a multivariable model, HIV-infected adults had a 30% lower odds of hypertension, compared to HIV-uninfected adults (aOR = 0.70, 95% CI: 0.57-0.85). In a separate multivariable model, HIV-infected adults with CD4 ≤200 cells/mm3 had a 44% lower odds of hypertension (aOR = 0.56, 95% CI: 0.38-0.83), as compared to adults with CD4 >200 cells/mm3. The mean arterial blood pressure was 6.5 mmHg higher among HIV-infected adults after HIV testing (p

BackgroundLysine acetylation is a crucial type of protein post-translational modification, which is involved in many important cellular processes and serious diseases. However, identification of protein acetylated sites through traditional experiment methods is time-consuming and laborious. Those methods are not suitable to identify a large number of acetylated sites quickly. Therefore, computational methods are still very valuable to accelerate lysine acetylated site finding.ResultIn this study, many biological characteristics of acetylated sites have been investigated, such as the amino acid sequence around the acetylated sites, the physicochemical property of the amino acids and the transition probability of adjacent amino acids. A logistic regression method was then utilized to integrate these information for generating a novel lysine acetylation prediction system named LAceP. When compared with existing methods, LAceP overwhelms most of state-of-the-art methods. Especially, LAceP has a more balanced prediction capability for positive and negative datasets.ConclusionLAceP can integrate different biological features to predict lysine acetylation with high accuracy. An online web server is freely available at http://www.scbit.org/iPTM/.

Online messaging app Telegram has increased in popularity in recent years surpassing Twitter and Snapchat by the number of active monthly users in late 2020. The messenger has also been crucial to protest movements in several countries in 2019-2020, including Belarus, Russia and Hong Kong. Yet, to date only few studies examined online activities on Telegram and none have analyzed the platform with regard to the protest mobilization. In the present study, we address the existing gap by examining Telegram-based activities related to the 2019 protests in Hong Kong. With this paper we aim to provide an example of methodological tools that can be used to study protest mobilization and coordination on Telegram. We also contribute to the research on computational text analysis in Cantonese-one of the low-resource Asian languages,-as well as to the scholarship on Hong Kong protests and research on social media-based protest mobilization in general. For that, we rely on the data collected through Telegram's API and a combination of network analysis and computational text analysis. We find that the Telegram-based network was cohesive ensuring efficient spread of protest-related information. Content spread through Telegram predominantly concerned discussions of future actions and protest-related on-site information (i.e., police presence in certain areas). We find that the Telegram network was dominated by different actors each month of the observation suggesting the absence of one single leader. Further, traditional protest leaders-those prominent during the 2014 Umbrella Movement,-such as media and civic organisations were less prominent in the network than local communities. Finally, we observe a cooldown in the level of Telegram activity after the enactment of the harsh National Security Law in July 2020. Further investigation is necessary to assess the persistence of this effect in a long-term perspective.

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity during adolescence, leading to altered postural control with compromised stability. To identify the effects of AIS on whole-body balance control during obstacle-crossing, 14 adolescents with Lenke 1 thoracic AIS and 14 healthy controls were compared in terms of the inclination angle (IA) of the body's center of mass (COM) relative to the center of pressure (COP), the rate of change of IA (RCIA) and the jerk index of IA. Between-side comparisons were also performed for the AIS group. The patients showed less smooth COM-COP motion in the sagittal plane with significantly increased anterior RCIA and IA jerk index during crossing with either the concave side (p = 0.001) or the convex side (p = 0.001) leading when compared to healthy controls. In the frontal plane, the patients showed close-to-zero RCIA (p = 0.002) while crossing with the leading limb, with an increased IA magnitude (p = 0.039) only while crossing with the concave-side limb leading. The patients with Lenke 1 thoracic AIS were found to cross obstacles with altered, compromised COM-COP control in both sagittal and frontal planes when compared to healthy controls. The results suggest that the thoracic spinal deformity in Lenke 1 AIS affects the whole-body balance control during obstacle-crossing, which should be monitored for signs of increased risk of loss of balance in the management of such patient groups.

INTRODUCTION:Altered trunk shape and body alignment in Lenke 1 thoracic adolescent idiopathic scoliosis (AIS) may affect the body's balance control during activities. The current study aimed to identify the effects of Lenke 1 thoracic AIS on the balance control during level walking in terms of the inclination angles (IA) of the center of mass (COM) relative to the center of pressure (COP), the rate of change of IA (RCIA), and the jerk index of IA. The association between the Cobb angle, IA and RCIA was also evaluated. MATERIALS AND METHODS:Sixteen adolescents with AIS (age: 14.0±1.8 years, height: 154.8±4.7 cm, mass: 42.0±7.5 kg) and sixteen healthy controls (age: 14.4±2.0 years, height: 158.4±6.2 cm, body mass: 48.6±8.9 kg) performed level walking in a gait laboratory. The kinematic and ground reaction force data were measured for both concave-side and convex-side limb cycles, and used to calculate the IA and RCIA, the jerk index of IA, and the temporal-spatial parameters. Correlations between the Cobb angle, IA and RCIA were quantified using Pearson's correlation coefficients (r). RESULTS:The patients showed less smooth COM-COP motion with increased jerk index of IA in the sagittal plane during single limb support (SLS) of the concave-limb (p = 0.05) and in the frontal plane during double limb support (DLS) (p < 0.05). The patients also showed significantly increased posterior RCIA on both the concave and convex side during initial (p = 0.04, p = 0.03) and terminal (p = 0.04, p = 0.03) DLS when compared to healthy controls. In the frontal plane, the patients walking on the concave-side limb showed decreased IA over SLS (p = 0.01), and at contralateral toe-off (p

OBJECTIVE: The objective of this study was to evaluate the relationship between the trabecular bone microarchitecture and cortical bone morphology by using micro-computed tomography (micro-CT) and dental cone-beam computed tomography (dental CT). MATERIALS AND METHODS: Sixteen femurs and eight fifth lumbar vertebrae were collected from eight male Sprague Dawley rats. Four trabecular bone microarchitecture parameters related to the fifth lumbar vertebral body (percent bone volume [BV/TV], trabecular thickness [TbTh], trabecular separation [TbSp], and trabecular number [TbN]) were calculated using micro-CT. In addition, the volumetric cancellous bone grayscale value (vCanGrayscale) of the fifth lumbar vertebral body was measured using dental CT. Furthermore, four cortical bone morphology parameters of the femoral diaphysis (total cross-sectional area [TtAr], cortical area [CtAr], cortical bone area fraction [CtAr/TtAr], and cortical thickness [CtTh]) were calculated using both micro-CT and dental CT. Pearson analysis was conducted to calculate the correlation coefficients (r) of the micro-CT and dental CT measurements. Paired-sample t tests were used to compare the differences between the measurements of the four cortical bone morphology parameters obtained using micro-CT and dental CT. RESULTS: High correlations between the vCanGrayscale measured using dental CT and the trabecular bone microarchitecture parameters (BV/TV [r = 0.84] and TbTh [r = 0.84]) measured using micro-CT were observed. The absolute value of the four cortical bone morphology parameters may be different between the dental CT and micro-CT approaches. However, high correlations (r ranged from 0.71 to 0.90) among these four cortical bone morphology parameters measured using the two approaches were obtained. CONCLUSION: We observed high correlations between the vCanGrayscale measured using dental CT and the trabecular bone microarchitecture parameters (BV/TV and TbTh) measured using micro-CT, in addition to high correlations between the cortical bone morphology measured using micro-CT and dental CT. Further experiments are necessary to validate the use of dental CT on human bone.

It has been proposed that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) can reduce the risk of ventricular arrhythmias in post-MI patients. Abnormal Ca(2+) handling has been implicated in the genesis of post-MI ventricular arrhythmias. Therefore, we tested the hypothesis that dietary n-3 PUFAs alter the vulnerability of ventricular myocytes to cellular arrhythmia by stabilizing intracellular Ca(2+) cycling. To test this hypothesis, we used a canine model of post-MI ventricular fibrillation (VF) and assigned the animals to either placebo (1 g/day corn oil) or n-3 PUFAs (1-4 g/day) groups. Using Ca(2+) imaging techniques, we examined the intracellular Ca(2+) handling in myocytes isolated from post-MI hearts resistant (VF-) and susceptible (VF+) to VF. Frequency of occurrence of diastolic Ca(2+) waves (DCWs) in VF+ myocytes from placebo group was significantly higher than in placebo-treated VF- myocytes. n-3 PUFA treatment did not decrease frequency of DCWs in VF+ myocytes. In contrast, VF- myocytes from the n-3 PUFA group had a significantly higher frequency of DCWs than myocytes from the placebo group. In addition, n-3 PUFA treatment increased beat-to-beat alterations in the amplitude of Ca(2+) transients (Ca(2+) alternans) in VF- myocytes. These n-3 PUFAs effects in VF- myocytes were associated with an increased Ca(2+) spark frequency and reduced sarcoplasmic reticulum Ca(2+) content, indicative of increased activity of ryanodine receptors. Thus, dietary n-3 PUFAs do not alleviate intracellular Ca(2+) cycling remodeling in myocytes isolated from post-MI VF+ hearts. Furthermore, dietary n-3 PUFAs increase vulnerability of ventricular myocytes to cellular arrhythmia in post-MI VF- hearts by destabilizing intracellular Ca(2+) handling.

STIM1 is a Ca(2+) sensor within the ER membrane known to activate the plasma membrane store-operated Ca(2+) channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca(2+) signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca(2+) sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies.

Nitric oxide (NO) and superoxide (O(2) (-)) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2) (.-) must exist at defined levels. Unfortunately, the NO and O(2) (.-) levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O(2) (.-) while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1(-/-)) mice, a known model of NO/O(2) (.-) imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2) (.-) (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1(-/-) myocytes. Furthermore, EMEPO increased NOS1(-/-) myocyte basal contraction (Ca(2+) transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca(2+) leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1(-/-) myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O(2) (.-) levels may have therapeutic potential in the treatment of various cardiomyopathies.

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.

BACKGROUND: Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met. METHODOLOGY/PRINCIPAL FINDINGS: Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA. CONCLUSION/SIGNIFICANCE: The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.

BACKGROUND: Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274-281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization. CONCLUSIONS/SIGNIFICANCE: The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.

BACKGROUND: Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. METHODS/PRINCIPAL FINDINGS: We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. CONCLUSIONS/SIGNIFICANCE: Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.