Compositional features analysis by machine learning in genome represents linear adaptation of monkeypox virus.

Introduction: The global headlines have been dominated by the sudden and widespread outbreak of monkeypox, a rare and endemic zoonotic disease caused by the monkeypox virus (MPXV). Genomic composition based machine learning (ML) methods have recently shown promise in identifying host adaptability and evolutionary patterns of virus. Our study aimed to analyze the genomic characteristics and evolutionary patterns of MPXV using ML methods. Methods: The open reading frame (ORF) regions of full-length MPXV genomes were filtered and 165 ORFs were selected as clusters with the highest homology. Unsupervised machine learning methods of t-distributed stochastic neighbor embedding (t-SNE), Principal Component Analysis (PCA), and hierarchical clustering were performed to observe the DCR characteristics of the selected ORF clusters. Results: The results showed that MPXV sequences post-2022 showed an obvious linear adaptive evolution, indicating that it has become more adapted to the human host after accumulating mutations. For further accurate analysis, the ORF regions with larger variations were filtered out based on the ranking of homology difference to narrow down the key ORF clusters, which drew the same conclusion of linear adaptability. Then key differential protein structures were predicted by AlphaFold 2, which meant that difference in main domains might be one of the internal reasons for linear adaptive evolution. Discussion: Understanding the process of linear adaptation is critical in the constant evolutionary struggle between viruses and their hosts, playing a significant role in crafting effective measures to tackle viral diseases. Therefore, the present study provides valuable insights into the evolutionary patterns of the MPXV in 2022 from the perspective of genomic composition characteristics analysis through ML methods.

Convolutional Neural Networks Based on Sequential Spike Predict the High Human Adaptation of SARS-CoV-2 Omicron Variants.

The COVID-19 pandemic has frequently produced more highly transmissible SARS-CoV-2 variants, such as Omicron, which has produced sublineages. It is a challenge to tell apart high-risk Omicron sublineages and other lineages of SARS-CoV-2 variants. We aimed to build a fine-grained deep learning (DL) model to assess SARS-CoV-2 transmissibility, updating our former coarse-grained model, with the training/validating data of early-stage SARS-CoV-2 variants and based on sequential Spike samples. Sequential amino acid (AA) frequency was decomposed into serially and slidingly windowed fragments in Spike. Unsupervised machine learning approaches were performed to observe the distribution in sequential AA frequency and then a supervised Convolutional Neural Network (CNN) was built with three adaptation labels to predict the human adaptation of Omicron variants in sublineages. Results indicated clear inter-lineage separation and intra-lineage clustering for SARS-CoV-2 variants in the decomposed sequential AAs. Accurate classification by the predictor was validated for the variants with different adaptations. Higher adaptation for the BA.2 sublineage and middle-level adaptation for the BA.1/BA.1.1 sublineages were predicted for Omicron variants. Summarily, the Omicron BA.2 sublineage is more adaptive than BA.1/BA.1.1 and has spread more rapidly, particularly in Europe. The fine-grained adaptation DL model works well for the timely assessment of the transmissibility of SARS-CoV-2 variants, facilitating the control of emerging SARS-CoV-2 variants.

Penfluridol targets acid sphingomyelinase to inhibit TNF signaling and is therapeutic against inflammatory autoimmune diseases.

BACKGROUND: Penfluridol, isolated from an FDA-approved small-molecule drug library as an inhibitor of tumor necrosis factor α (TNFα)-stimulated NF-κB activation, is clinically used to treat chronic schizophrenia and related disorders. This study is aimed to investigate the therapeutic effect of penfluridol on TNFα-stimulated inflammatory autoimmune diseases, particularly inflammatory arthritis. METHODS: Various in vitro studies to confirm the inhibitory effect of penfluridol on TNFα-induced NF-κB activity in bone marrow-derived macrophages or Raw 264.7 macrophage cell line. In vivo studies assessed the therapeutic effects of penfluridol in various disease models, including TNFα transgenic mice, collagen-induced arthritis, DSS-induced colitis, and TNBS-induced colitis. Identification and characterization of the binding of penfluridol to acid sphingomyelinase using bioinformatics and drug affinity responsive target stability assay. Acid sphingomyelinase activity assays to reveal penfluridol-mediated inhibition of acid sphingomyelinase activity. siRNA knockdown experiments to illustrate the dependence of penfluridol's anti-TNF activity on acid sphingomyelinase. RESULTS: Penfluridol effectively inhibited TNFα-induced NF-κB activation in vitro and alleviated the severity of arthritis and colitis in vivo. Mechanistic studies revealed that penfluridol bound to acid sphingomyelinase and inhibited its activation. In addition, knockdown of acid sphingomyelinase largely abolished the inhibitory effects of penfluridol on TNFα-induced inflammatory cytokine production. Furthermore, penfluridol suppressed the differentiation of spleen naive CD4+T cells to TH1 and TH17 and inhibited M1 macrophage polarization. CONCLUSION: This study provides the rationale for the possible innovative use of penfluridol as a newly identified small-molecule drug for TNFα-driven diseases, such as inflammatory arthritis and colitis.

Generation and Characterization of a Nanobody Against SARS-CoV.

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

The safety of esophagojejunostomy via a transorally inserted-anvil method vs extracorporeal anastomosis using a circular stapler during total gastrectomy for Siewert type 2 adenocarcinoma of the esophagogastric junction.

BACKGROUND: Intracorporeal esophagojejunostomy via a transorally inserted-anvil method during laparoscopic total gastrectomy (LTG) for upper gastric cancer has been demonstrated to be feasible, but the use of this assessment exclusively for Siewert type 2 adenocarcinoma of the esophagogastric junction (AEG) has not been reported. METHODS: A total of 428 consecutive gastric-cancer patients who underwent LTG in Nanfang Hospital from January 2008 to December 2016 were reviewed. Among these patients, 98 were classified as Siewert type 2 AEG. The patients underwent intracorporeal esophagojejunostomy through either a transorally inserted-anvil method (n = 27) or extracorporeal anastomosis usinga circular stapler (n = 71). After generating propensity scores with covariates that were associated with developing anastomotic leakage, 26 patients who underwent esophagojejunostomy via the transorally inserted-anvil method (transoral group) were 1:1 matched with 26 patients who underwent the procedure via extracorporeal anastomosis using a circular stapler (extracorporeal group). The safety after 30 days post-operatively was compared between the two groups. RESULTS: The transoral group and extracorporeal group were balanced regarding the baseline variables. The operative time, reconstruction duration, number of dissected lymph nodes, length of the proximal resection margins, estimated blood loss, intra-operative complication rate, and post-operative recovery course were not significantly different between the two groups. The mean anvil-insertion completion time (9.7 ± 3.0 vs 13.4 ± 2.0 minutes, P < 0.001) and the median incision length (5.5 vs 7.0 cm, P < 0.001) in the transoral group were shorter than those in the extracorporeal group. The incidence of post-operative complications (26.9% vs 23.1%, P = 0.749) and the classification of complication severity (P = 0.939) were similar between the two groups. CONCLUSIONS: Intracorporeal esophagojejunostomy through a transorally inserted-anvil method may be a potentially safe approach to simplify and optimize the procedure during LTG for Siewert type 2 AEG.

EPHA2 feedback activation limits the response to PDEδ inhibition in KRAS-dependent cancer cells.

KRAS is one of the most important proto-oncogenes. Its mutations occur in almost all tumor types, and KRAS mutant cancer is still lack of effective therapy. Prenyl-binding protein phosphodiesterase-δ (PDEδ) is required for the plasma membrane association and subsequent activation of KRAS oncogenic signaling. Recently, targeting PDEδ has provided new promise for KRAS mutant tumors. However, the therapeutic potential of PDEδ inhibition remains obscure. In this study, we explored how PDEδ inhibition was responded in KRAS mutant cancer cells, and identified KRAS mutant subset responsive to PDEδ inhibition. We first performed siRNA screen of KRAS growth dependency of a small panel of human cancer lines, and identified a subset of KRAS mutant cancer cells that were highly dependent on KRAS signaling. Among these cells, only a fraction of KRAS-dependent cells responded to PDEδ depletion, though KRAS plasma membrane association was effectively impaired. We revealed that the persistent RAF/MEK/ERK signaling seemed responsible for the lack of response to PDEδ depletion. A kinase array further identified that the feedback activation of EPH receptor A2 (EPHA2) accounted for the compensatory activation of RAF/MEK/ERK signaling in these cells. Simultaneous inhibition of EPHA2 and PDEδ led to the growth inhibition of KRAS mutant cancer cells. Together, this study gains a better understanding of PDEδ-targeted therapeutic strategy and suggests the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.

Microcatheter "First-Pass Effect" Predicts Acute Intracranial Artery Atherosclerotic Disease-Related Occlusion.

BACKGROUND: The differentiation between intracranial atherosclerotic stenosis (ICAS) and intracranial embolism as the immediate cause of acute ischemic stroke requiring endovascular therapy is important but challenging. In cases of ICAS, we often observe a phenomenon we call the microcatheter "first-pass effect," which is temporary blood flow through the occluded intracranial artery when the angiographic microcatheter is initially advanced through the site of total occlusion and immediately retrieved proximally. OBJECTIVE: To evaluate whether this microcatheter first-pass effect can be used to differentiate ICAS from intracranial embolism. METHODS: A total of 61 patients with acute ischemic stroke resulting from large intracranial artery occlusion and in whom recanalization was achieved by endovascular treatment were included in the study. The microcatheter first-pass effect was tested in these patients. The sensitivity, specificity, positive predictive values (PPV), and accuracy of the microcatheter first-pass effect for prediction of ICAS were assessed. RESULTS: The microcatheter first-pass effect was more frequently observed in patients with ICAS than in those with intracranial embolism (90.9% vs 12.8%, P < .001). For identifying ICAS, sensitivity, specificity, PPV, and accuracy of the microcatheter first-pass effect were 90.9%, 87.2%, 80.0%, 88.5%, respectively. CONCLUSION: The sensitivity and PPV of the microcatheter first-pass effect are high for prediction of ICAS in patients with acute symptoms.

Special Endovascular Treatment for Acute Large Artery Occlusion Resulting From Atherosclerotic Disease.

BACKGROUND: Acute intracranial atherosclerotic disease (IAD)-related large artery occlusion (LAO) is typically refractory to mechanical thrombectomy. We evaluated the feasibility and safety of emergency balloon-assisted or stent-assisted angioplasty performed with tirofiban administration for acute IAD-related LAO. METHODS: We identified, from among 55 consecutive patients who underwent endovascular treatment for LAO, 12 patients with acute IAD-related LAO who underwent balloon-assisted or stent-assisted angioplasty with (n = 3) or without passage of a stent retriever. The treatment included tirofiban administration. We obtained, from patients' clinical records, thrombolysis in cerebral infarction scores (to assess the extent of reperfusion), follow-up magnetic resonance angiography images (to assess patency of the responsive arteries), and 90-day modified Rankin (mRS) scores (to assess outcomes). RESULTS: Temporary blood flow and severe stenosis were observed angiographically in all 12 patients, either when the stent retriever was deployed or when a microcatheter was advanced through the site of occlusion. Persistent recanalization was achieved in all patients, and there was no operative complication or arterial reocclusion. All 8 patients with an occluded major artery in the anterior circulation had a good outcome, with an mRS score of ≤2. Two of the 4 patients with basilar artery occlusion had a good outcome, with an mRS score of ≤2. One patient (25%) died within 72 hours after procedure. CONCLUSIONS: Our data point to the safety and feasibility of emergency balloon-assisted or stent-assisted angioplasty performed with tirofiban administration and a single or no passage of the stent retriever for acute IAD-related LAO.

Diagnostic ability of 3-dimensional contrast-enhanced MR angiography in identifying vertebral basilar artery stenosis.

BACKGROUND: Vertebral-basilar artery stenosis is associated with posterior circulation infarction. So correct detection of vertebral basilar artery stenosis is very important. Studies concerning the sensitivity and specificity of 3-dimensional contrast enhanced MR angiography (3D-CE-MRA) in detecting vertebral basilar artery stenosis is generally lacking. METHODS: Retrospectively reviewed the imagines of consecutive one hundred and forty-nine Chinese patients with ischemic stroke or vertigo/dizziness who underwent 3D-CE-MRA and DSA. DSA and CE-MRA images were studied separately and to determine the presence of mild, moderate, or severe stenosis of the vertebral-basilar arteries. Analysis combined with vascular origin image was applied when evaluating the vertebral artery origin stenosis. Sensitivity, specificity, positive and negative predictive values, and the accuracy of 3D-CE-MRA in detecting and grading of vertebral-basilar artery stenosis were calculated. RESULTS: Compared with DSA, sensitivity, specificity and accuracy of 3D-CE-MRA in detecting of vertebral artery origin ≥70% stenosis or occlusion was 97.1%, 77.4% and 81.9%, but diagnostic consistency was poor (K=0.59); Analysis combined with vascular origin images, the specificity (97.8%), accuracy (92.9%) and consistency (K=0.826) was significantly improved. CONCLUSIONS: 3D-CE-MRA is a sensitive and noninvasive technique for the detection of vertebral artery origin stenosis. Furthermore, analysis combined with vascular origin image would improve the diagnostic accuracy.

Effects of sulfonylureas on lipids in type 2 diabetes mellitus: a meta-analysis of randomized controlled trials.

OBJECTIVE: Previous studies suggested that dyslipidemia was potentially associated with anti-diabetic medications of sulfonylureas (SUs). The results were, however, inconsistent. Therefore, we conducted a meta-analysis of randomized controlled trials (RCTs) to assess the effects of SUs on the level of lipids in patients with type 2 diabetes mellitus (T2DM). METHODS: We searched PubMed, EMBASE, and CENTRAL databases for RCTs that addressed the effects of second- and/or third-generation SUs used in T2DM patients on lipids profiles with study duration of at least 12 weeks. Two reviewers independently screened literature, collected data, and assessed methodological quality of included studies. The meta-analysis was performed by using the RevMan5.1 software. RESULTS: A total of 59 RCTs were included, of which 52 were included for final meta-analysis. The results suggested that SUs statistically increased the levels of FFA (SMD = 0.24, 95%CI 0.06 to 0.42) and TG (MD = 0.06, 95%CI 0.02 to 0.10), but decreased HDL-C (MD = -0.07, 95%CI -0.11 to -0.04) and LDL-C (MD = -0.11, 95%CI -0.17 to -0.04); but the SUs had no effect on TC (MD = 0.01, 95%CI -0.05 to 0.08), ApoA1 (MD = 0.01, 95%CI -0.03 to 0.04), and Apo B (MD = -0.01, 95%CI -0.05 to 0.03). When compared to metformin, SUs could increase TC and LDL-C; compared to glinides, SUs increased TC and lowered HDL-C; compared to thiazolidinediones, SUs reduced TC, LDL-C, HDL-C, and increase TG. CONCLUSIONS: SUs have a small effect on lipids, although they may statistically increase the level of FFA and TG, and decrease LDL-C and HDL-C.

Value of fused positron emission tomography CT in detecting primaries in patients with primary unknown cervical lymph node metastasis.

OBJECTIVE: Identification of the primary tumour can prolong the life expectancy of patients with primary unknown cervical lymph node metastasis (PUCLNM) through targeted therapy. This study investigated the value of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET-CT) at identifying primaries in patients with PUCLNM. METHODS: Twenty-seven patients (21 males and 6 females, median age 48.2 ± 16.3, age range 30-73) with PUCLNM underwent FDG PET-CT to search for the primary tumour, which could not be detected by conventional diagnostic modalities. The results were analysed and correlated with either pathological findings or clinical follow up. RESULTS: Pathological FDG uptake suspicious for the primary was detected in 13 cases, while the primary tumour remained occult in 14 cases. Eleven of 13 patients with suspected primaries were confirmed by histological findings. One with a coexisting second tumour and three with unexpected distant metastases were found in patients with confirmed primaries. The most common primary location in patients with PUCLNM found in our study was nasopharynx. In those 14 patients with negative FDG PET-CT results, only one patient had a primary malignancy that was proven histologically after endoscopy with biopsy during a period of clinical follow up. The sensitivity, specificity, accuracy and positive predictive values of FDG PET-CT were 91.7, 86.7, 88.9 and 84.6%, respectively. CONCLUSION: FDG PET-CT is a useful tool to help search for unknown primaries in patients with cervical lymph node metastasis and has an acceptable diagnostic yield for the detection of distant malignancies.

Treatment of recurrent head and neck carcinoma involving the carotid artery: carotid reconstruction with ePTFE graft.

The aim of our study is to investigate the feasibility of reconstructing the carotid artery using expanded polytetraflouroethylene (ePTFE) in patients with recurrent head and neck carcinoma involving the carotid artery. Ten patients, who had recurrent head and neck carcinoma involving the carotid artery, received carotid artery resection and reconstruction with ePTFE, tissue defects were repaired by pectoralis major myocutaneous flap. Results show that eight patients did not present any vascular and neurologic complications. One patient presented slight hemiparesis, another patient developed wound infection and pharyngocutaneous fistula. The mean follow-up period was 33.1 ± 16.0 months. The 2-year survival rate was 50% (5/10), and there was one patient who survived for 60 months without locoreginal recurrence or distant metastasis. En bloc resection of tumor and involved carotid-associated ePTFE reconstruction provide effective improvement in the locoregional control of the recurrent head and neck carcinoma. The pedicle pectoralis major myocutaneous flap can provide not only wound bed with affluent blood supply for the vascular grafts, but also reparation of skin or the tissue defects of oropharynx and hypopharynx.

[Effects of combining tumor necrosis factor related apoptosis-inducing ligand with PI3-K-Akt inhibition on nasopharyngeal carcinoma cell].

OBJECTIVE: To study the effects of combinative therapy of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and PI3-K-Akt inhibitor on the growth and apoptosis of nasopharyngeal carcinoma (NPC) cells and underlying mechanisms. METHODS: With cell growth assay, flow cytometric analysis and Western blotting, the effects of TRAIL and PI3-K-Akt special inhibitor (LY294002) on cell growth, apoptosis and related proteins expressions in CNE-2 cell lines were studied. RESULTS: When concentrate of TRAIL>1 ng/ml, viability rate of cells in combinative treatment group with TRAIL and LY294002 was higher than that in the single treatment group with TRAIL (all P<0.05). When concentrate of TRAIL were 10 ng/ml and 100 ng/ml, the combinative treatment induced CNE-2 apoptosis more obviously than single treatments (t were 7.167 and 7.206, all P<0.05). The combination group showed more cleavage of Caspase-8, Caspase-3, Caspase-9 than single treatment groups. CONCLUSIONS: Combinative application of TRAIL and PI3-K-Akt pathway inhibitor inhibits the growth of CNE-2 and induces apoptosis. The mitochondrial dependent pathway is implicated for the underlying mechanism.

Simultaneously targeting Bcl-2 and Akt pathways reverses resistance of nasopharyngeal carcinoma to TRAIL synergistically.

AIMS AND BACKGROUND: Despite progress in treatment techniques, the five-year survival rate of nasopharyngeal carcinoma (NPC) is disappointing. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. Given the antiapoptotic functions of Bcl-2 and Akt, we examined the effects of targeting these pathways alone or simultaneously on TRAIL apoptosis in NPC cell lines. METHODS AND STUDY DESIGN: We first tested the cytotoxic effect of TRAIL and the expression of death receptors, Bcl-2, Akt, and p-Akt on four NPC cell lines by MTT and Western blotting, respectively. Small interfering RNAs (siRNAs) targeting Bcl-2 and PI3-K inhibitor (LY294002) were used alone or combined with TRAIL in the cell lines and cytotoxicity was examined by MTT. Apoptosis rates, mitochondrial transmembrane potential, and apoptotic pathway signals were detected by flow cytometric analysis, DiOC6(3) assays, and Western blotting after the various combination treatments on CNE-2, the cell line that was most resistant to TRAIL. RESULTS: Although no direct correlation between the sensitivity to TRAIL and the relative expression levels of Bcl-2 and activated Akt was found in the NPC cell lines examined, siRNA mediated the downregulation of Bcl-2 and LY294002-induced inactivation of Akt, increasing the sensitivity of all examined NPC cell lines to TRAIL. Synergistic enhancement of TRAIL-mediated cytotoxicity was observed in combination treatment of Bcl-2 siRNA and LY294002 compared to cells treated with each treatment alone. The synergistic effects were mediated through increased apoptotic signaling of the mitochondrial pathway, as was evident from the more increased mitochondrial depolarization, activation of caspase-9 and caspase-3, and suppression of XIAP. CONCLUSIONS: This study provides proof of principle that TRAIL combined with simultaneously targeting the Bcl-2 and Akt signaling pathways may have potential as a novel future treatment strategy for NPC.

[Projecting from the superior olivary complex to the inner ear in the cat: a retrograde fluorescent labelling study].

OBJECTIVE: To investigate the distribution and projective feature of cat olivocochlear neurons. METHODS: Eleven adult cats were divided into two groups randomly. The experimental group of eight cats was injected of 1% cholera toxin B (CTB) to the left cochlea, while injected of 5% fluoro gold (FG) to the right cochlea. The control group of three cats was injected of saline to bilateral cochlea. After a survival time of 7 days, serial frozen sections were cut in the cat brainstem. All the sections were processed by immunofluorescent procedure for CTB and FG, and the labeled olivocochlear neurons were observed by fluorescent microscope. RESULTS: In the experimental group, the mean total of olivocochlear neurons labeled by CTB and FG was 3210 +/- 168, including lateral olivocochlear neurons (LOC, 2298 +/- 120) and medial olivocochlear neurons (MOC, 913 +/- 64). The labeled neurons were divided into three different types according to their feature of projection: neurons which only projected to the ipsilateral cochlea, neurons which only projected to the contralateral cochlea, and double-labeled neurons which projected both to the ipsilateral and contralateral cochlea, but the double-labeled neurons comprised 3.9% and 15.1% in the LOC and MOC system respectively. No labeled neurons were found in the control group. CONCLUSIONS: There are three types of neurons in the cat olivocochlear system. The neurons which projected to the bilateral cochlea may distribute both in the LOC and MOC system.