In this study, combinatorial libraries were used in conjunction with ultra-high throughput sequencing to comprehensively determine the impact of each of the 19 possible amino acid substitutions at each residue position in the TEM-1β-lactamase enzyme. The libraries were introduced into E. coli and mutants were selected for ampicillin resistance. The selected colonies were pooled and subjected to ultra-high throughput sequencing to reveal the sequence preferences at each position. The depth of sequencing provided a clear, statistically significant picture of what amino acids are favored for ampicillin hydrolysis for all 263 positions of the enzyme in one experiment. Although the enzyme is generally tolerant of amino acid substitutions, several surface positions far from the active site are sensitive to substitutions suggesting a role for these residues in enzyme stability, solubility or catalysis. In addition, information on the frequency of substitutions was used to identify mutations that increase enzyme thermodynamic stability. Finally, a comparison of sequence requirements based on the mutagenesis results versus those inferred from sequence conservation in an alignment of 156 class A β-lactamases reveals significant differences in that several residues in TEM-1 do not tolerate substitutions and yet extensive variation is observed in the alignment, and vice versa. An analysis of the TEM-1 and other class A structures suggests residues that vary in the alignment may nevertheless make unique, but important, interactions within individual enzymes.

Background: MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that regulate gene expression and function, but little is known about regulation of miRNAs in the kidneys under normal or pathologic conditions. Here, we sought to investigate the potential involvement of miRNAs in renal ischemia/reperfusion (I/R) injury and angiogenesis and to define some of the miRNAs possibly associated with renal angiogenesis. Methods and Results: Male Balb/c mice were subjected to a standard renal I/R. CD31 immunostaining indicated a significant increase of microvessels in the ischemic region. VEGF and VEGFR2 expression were increased in renal I/R at both the mRNA and protein levels which were detected by qRT-PCR and Western blot, respectively. More importantly, 76 microRNAs exhibited more than 2-fold changes using Agilent microRNA microarray, which contains downregulation of 40 miRNAs and upregulation of 36 miRNAs. Upregulation of miR-210 was confirmed by qRT-PCR with prominent changes at 4 and 24 h after reperfusion. Furthermore, overexpression of miR-210 in HUVEC-12 cells enhances VEGF and VEGFR2 expression and promotes angiogenesis on Matrigel in vitro. Conclusion: These findings suggest miR-210 may be involved in targeting the VEGF signaling pathway to regulate angiogenesis after renal I/R injury, which provides novel insights into the angiogenesis mechanism of renal I/R injury.

Angiogenesis after ischemic brain injury contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Recent researches have demonstrated that dysfunction of long non-coding RNAs are associated with angiogenesis. We have previously reported that long non-coding RNAs (lncRNAs) are aberrantly expressed in ischemic stroke. However, little is known about long non-coding RNAs and theirs role in angiogenesis after stroke. In this study, we identified a rat lncRNAs, Meg3, and found that Meg3 was significantly decreased after ischemic stroke. Overexpression of Meg3 suppressed functional recovery and decreased capillary density after ischemic stroke. Downregulation of Meg3 ameliorated brain lesion and increased angiogenesis after ischemic stroke. Silencing of Meg3 resulted in a proangiogenic effect evidenced by increased endothelial cell migration, proliferation, sprouting, and tube formation. Mechanistically, we showed that Meg3 negatively regulated notch pathway both in vivo and in vitro. Inhibition of notch signaling in endothelial cells reversed the proangiogenic effect induced by Meg3 downregulation. This study revealed the function of Meg3 in ischemic stroke and elucidated its mechanism in angiogenesis after ischemic stroke.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-016-0270-z) contains supplementary material, which is available to authorized users.

Ischemic injuries will lead to necrotic tissue damage, and post-ischemia angiogenesis plays critical roles in blood flow restoration and tissue recovery. Recently, several types of small RNAs have been reported to be involved in this process. In this study, we first generated a rat brain ischemic model to investigate the involvement of new types of small RNAs in ischemia. We utilized deep sequencing and bioinformatics analyses to demonstrate that the level of small RNA fragments derived from tRNAs strikingly increased in the ischemic rat brain. Among these sequences, tRNAVal- and tRNAGly-derived small RNAs account for the most abundant segments. The up-regulation of tRNAVal- and tRNAGly-derived fragments was verified through northern blot and quantitative PCR analyses. The levels of these two fragments also increased in a mouse hindlimb ischemia model and cellular hypoxia model. Importantly, up-regulation of the tRNAVal- and tRNAGly-derived fragments in endothelial cells inhibited cell proliferation, migration and tube formation. Furthermore, we showed that these small RNAs are generated by angiogenin cleavage. Our results indicate that tRNA-derived fragments are involved in tissue ischemia, and we demonstrate for the first time that tRNAVal- and tRNAGly-derived fragments inhibit angiogenesis by modulating the function of endothelial cells.

Introduction: Coronavirus disease 2019 , caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread widely. The aim of this study was to investigate the dynamic changes in peripheral blood lymphocyte subsets in adult patients with COVID-19. Methods: The electronic medical records were reviewed. Data including demographic characteristics, clinical manifestations, comorbidities, laboratory data, and radiological examinations of 435 hospitalized COVID-19 patients with a confirmed SARS-CoV-2 viral infection were extracted and analyzed retrospectively. Lymphocyte subset counts at each week after the onset of the illness were compared with those of the other weeks of illness and with those of control individuals. Results: The various lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, and CD16/56+) were below the normal ranges at 1 week after the onset of illness, reaching a nadir during the second week. They increased gradually during the third week and returned to normal levels in the fifth week, but were still lower than those of the healthy controls. The CD3+, CD4+, and CD8+ counts were significantly lower in patients with severe disease compared to those with non-severe disease, and in patients who died compared to those who recovered. Discussion: This research indicates that the levels of peripheral blood lymphocyte subsets (CD3+, CD4+, and CD8+) are associated with disease progression and severity, and with the prognosis in patients with COVID-19. Dynamic monitoring of human immune function is one of the indicators for evaluating the severity of disease and the prognosis of COVID-19 patients, and is useful for formulating appropriate treatment strategies.

Highlights We investigated the prevalence and recovery time of olfactory and gustatory dysfunction in COVID-19 patients in Wuhan, China. This retrospective study of 196 patients with COVID-19 found that 8.2% and 4.6% of patients reported olfactory and taste disorders, respectively. Recovery from chemosensory dysfunction (OD and/or GD) was slow, with over half of the patients taking more than 4 weeks to recover. Patients with COVID-19 and olfactory and/or gustatory dysfunctions had significantly higher rates of cardiovascular disease than patients without olfactory and/or gustatory dysfunctions. The results indicate that the prevalence and prognosis of olfactory or gustatory impairment in COVID-19 patients vary globally, reflecting the complexity of this disease.

Posttraumatic hydrocephalus (PTH) is a disorder of disturbed cerebrospinal fluid (CSF) dynamics after traumatic brain injury (TBI). It can lead to brain metabolic impairment and dysfunction and has a high risk of clinical deterioration and worse outcomes. The incidence and risk factors for the development of PTH after decompressive craniectomy (DC) has been assessed in previous studies, but rare studies identify patients with higher risk for PTH among all TBI patients. This study aimed to develop and validate a risk scoring system to predict PTH after TBI. Demographics, injury severity, duration of coma, radiologic findings, and DC were evaluated to determine the independent predictors of PTH during hospitalization until 6 months following TBI through logistic regression analysis. A risk stratification system was created by assigning a number of points for each predictor and validated in an independent cohort. The model accuracy was assessed by the area under the receiver operating characteristic curve (AUC). Of 526 patients in the derivation cohort, 57 (10.84%) developed PTH during 6 months follow up. Age > 50 yrs (Odd ratio [OR] = 1.91, 95% confidence interval [CI] 1.09-3.75, 4 points), duration of coma ≥1 w (OR = 5.68, 95% CI 2.57-13.47, 9 points), Fisher grade III (OR = 2.19, 95% CI 1.24-4.36, 5 points) or IV (OR = 3.87, 95% CI 1.93-8.43, 7 points), bilateral DC (OR = 6.13, 95% CI 2.82-18.14, 9 points), and extra herniation after DC (OR = 2.36, 95% CI 1.46-4.92, 5 points) were independently associated with PTH. Rates of PTH for the low- (0-12 points), intermediate- (13-22 points) and high-risk (23-34 points) groups were 1.16%, 35.19% and 78.57% (p < 0.0001). The corresponding rates in the validation cohort, where 17/175 (9.71%) developed PTH, were 1.35%, 37.50% and 81.82% (p < 0.0001). The risk score model exhibited good-excellent discrimination in both cohorts, with AUC of 0.839 versus 0.894 (derivation versus validation) and good calibration (Hosmer-Lemshow p = 0.56 versus 0.68). This model will be useful to identify patients at high risk for PTH who may be candidates for preventive interventions, and to improve their outcomes.

Drug repurposing has become an alternative therapeutic strategy for cancer treatment given the known pharmacokinetics and toxicity. The inhibitory effects of artesunate have been reported in various cancers. In this work, we investigated the effects of artesunate in nasopharyngeal carcinoma (NPC). We demonstrate that artesunate significantly inhibits proliferation via arresting NPC cells at G2/M phase. It also induces apoptosis through caspase-dependent and mitochondria-independent pathways in multiple NPC cell lines. The combination of artesunate and cisplatin is synergistic in targeting NPC cells in in vitro cellular culture system and in vivo xenograft tumor models. Artesunate inhibits phosphorylation of essential molecules involved in Akt/mTOR pathway in NPC cells, such as Akt, mTOR, and 4EBP1, and its inhibitory effects are partially abolished by overexpression of constitutively active Akt. In addition, artesunate also induces mitochondrial dysfunction and oxidative stress via inhibiting mitochondrial respiration, increasing levels of mitochondrial superoxide and cellular reactive oxygen species (ROS), leading to decreased ATP levels. Two ROS scavengers partially abolish the inhibitory effects of artesunate in NPC cells. These data suggest that both inhibition of Akt/mTOR pathway and induction of ROS are required for the action of artesunate in NPC cells. Our work demonstrates that artesunate is a potential candidate for NPC treatment. Our work also highlights the critical roles of Akt/mTOR pathway and mitochondrial function in NPC cells.