Prevalence, toxin-genotype distribution, and transmission of Clostridium perfringens from the breeding and milking process of dairy farms.

This study aimed to elucidate the distribution, transmission, and cross-contamination of Clostridium perfringens during the breeding and milking process from dairy farms. The prevalence of 22.3% (301/1351) yielded 494 C. perfringens isolates; all isolates were type A, except for one type D, and 69.8% (345/494) of the isolates carried atyp. cpb2 and only 0.6% (3/494) of the isolates carried cons. cpb2. C. perfringens detected throughout the whole process but without type F. 150 isolates were classified into 94 pulsed-field gel electrophoresis (PFGE) genotypes; among them, six clusters contained 34 PFGE genotypes with 58.0% isolates which revealed epidemic correlation and genetic diversity; four PFGE genotypes (PT57, PT9, PT61, and PT8) were the predominant genotypes. The isolates from different farms demonstrated high homology. Our study confirmed that C. perfringens demonstrated broad cross-contamination from nipples and hides of dairy cattle, followed by personnel and tools and air-introduced raw milk during the milking process. In conclusion, raw milk could serve as a medium for the transmission of C. perfringens, which could result in human food poisoning. Monitoring and controlling several points of cross-contamination during the milking process are essential as is implementing stringent hygiene measures to prevent further spread and reduce the risk of C. perfringens infection.

Arterial Labeling Ultrasound Subtraction Angiography (ALUSA) Based on Acoustic Phase-Change Nanodroplets.

Biomedical imaging technology (like digital subtraction angiography (DSA)) based on contrast agents has been widely employed in the diagnosis of vascular-related diseases. While the DSA achieves the high-resolution observation of specified vessels and their downstream perfusion at the cost of invasive, radioactive operation and hepatorenal toxicity. To address those problems, this study develops arterial labeling ultrasound (US) subtraction angiography (ALUSA) based on a new perfluorobutane (PFB) nanodroplets with a lower vaporization threshold through spontaneous nucleation. The nanodroplets can be selectively vaporized to microbubbles, indicating a highly echogenic signal at B-mode images only using a diagnostic transducer. By labeling a single blood vessel for nanodroplets vaporization and tracking its downstream blood perfusion in segmental renal arteries at a frame rate of 500 Hz. The results demonstrate the color-coded super-resolution ALUSA image, exhibiting the downstream arcuate and interlobular arteries of each segmental renal artery with a resolution of 36 µm in a rabbit kidney. Furthermore, ALUSA could offer the vascular structures, blood flow velocity, and direction of their primary supply vessels in the mouse breast tumor. ALUSA fills the gap of noninvasive labeling angiography in US and opens a broad vista in the diagnosis and treatment of tumor and vascular-related diseases.

Anticancer Activity and Molecular Mechanism of Momordica cochinchinensis Seed Extract in Chronic Myeloid Leukemia Cells.

Targeting Bcr-Abl is the key to the treatment of chronic myeloid leukemia. Despite great progress in the treatment of patients with chronic CML, advanced CML patients are still unable to obtain effective and safe drugs. Momordica cochinchinensis seed is the dried ripe seed of Momordica cochinchinensis, which is a kind of fruit and consumed for dietary as well as medicinal uses. This study aimed to investigate the anticancer activity of Momordica cochinchinensis seed extract (MCSE) in CML cells. CML cells (KBM5 and KBM5-T315I) were treated with MCSE and analyzed for growth, apoptosis, and signal transduction. Nude mouse xenograft model was used to evaluate the antitumor activity of MCSE In Vivo. MCSE significantly reduced the cell viability of CML cells, triggered G0/G1 phase arrest in KBM5 cells and S phase arrest in KBM5-T315I cells. Concurrently, MCSE caused the activation of caspase-3, -8, -9, PARP and the degradation of Mcl-1, ultimately triggering endogenous and exogenous cell apoptosis. Meanwhile, MCSE downregulated Bcr-Abl levels and its downstream signaling pathways. Additionally, MCSE inhibited the growth of CML cells in nude mouse xenografts. Taken together, this study demonstrated the anticancer mechanism of MCSE, namely blocking Bcr-Abl and downregulating Mcl-1, and finally induced apoptosis of CML cells.

G3BP1 interacts with YWHAZ to regulate chemoresistance and predict adjuvant chemotherapy benefit in gastric cancer.

BACKGROUND: A large proportion of gastric cancer patients are susceptible to chemoresistance, while the underlying mechanism remains obscure. Stress granules (SGs) play a self-defence role for tumour cells in inhibiting chemotherapy-induced apoptosis. As an SG assembly effector, G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein) has been reported to be overexpressed in gastric cancer; thus, here we aim to explore its potent roles in gastric cancer chemoresistance. METHODS: Kaplan-Meier analysis was used to compare survival rates in gastric cancer patients with different G3BP1 expression. The influence of G3BP1 on gastric cancer cell chemoresistance and apoptosis were evaluated by in vitro and in vivo approaches. The interaction between G3BP1 and YWHAZ was assessed by immunohistochemistry, immunoprecipitation and immunofluorescence. RESULTS: G3BP1 was associated with the poor outcome of gastric cancer patients who received adjuvant chemotherapy. G3BP1 knockdown significantly increased the sensitivity of gastric cancer cells to chemotherapy drugs. Mechanically, cell apoptosis and pro-apoptotic-associated molecules were significantly elevated upon G3BP1 depletion. Gene co-expression network analyses identified YWHAZ as the critical interlayer of G3BP1; as a result, G3BP1 interacted with YWHAZ to sequester Bax into the cytoplasm. Clinically, G3BP1highYWHAZhigh gastric cancer patients displayed the worst outcome compared with other patients after chemotherapy. CONCLUSIONS: The expression of G3BP1 and YWHAZ could predict the adjuvant chemotherapy benefit in gastric cancer patients.

Pattern recognition of microcirculation with super-resolution ultrasound imaging provides markers for early tumor response to anti-angiogenic therapy.

Rationale: Cancer treatment outcome is traditionally evaluated by tumor volume change in clinics, while tumor microvascular heterogeneity reflecting tumor response has not been fully explored due to technical limitations. Methods: We introduce a new paradigm in super-resolution ultrasound imaging, termed pattern recognition of microcirculation (PARM), which identifies both hemodynamic and morphological patterns of tumor microcirculation hidden in spatio-temporal space trajectories of microbubbles. Results: PARM demonstrates the ability to distinguish different local blood flow velocities separated by a distance of 24 µm. Compared with traditional vascular parameters, PARM-derived heterogeneity parameters prove to be more sensitive to microvascular changes following anti-angiogenic therapy. Particularly, PARM-identified "sentinel" microvasculature, exhibiting evident structural changes as early as 24 hours after treatment initiation, correlates significantly with subsequent tumor volume changes (|r| > 0.9, P < 0.05). This provides prognostic insight into tumor response much earlier than clinical criteria. Conclusions: The ability of PARM to noninvasively quantify tumor vascular heterogeneity at the microvascular level may shed new light on early-stage assessment of cancer therapy.

Programmable ultrasound imaging guided theranostic nanodroplet destruction for precision therapy of breast cancer.

Ultrasound-stimulated contrast agents have gained significant attention in the field of tumor treatment as drug delivery systems. However, their limited drug-loading efficiency and the issue of bulky, imprecise release have resulted in inadequate drug concentrations at targeted tissues. Herein, we developed a highly efficient approach for doxorubicin (DOX) precise release at tumor site and real-time feedback via an integrated strategy of "programmable ultrasonic imaging guided accurate nanodroplet destruction for drug release" (PND). We synthesized DOX-loaded nanodroplets (DOX-NDs) with improved loading efficiency (15 %) and smaller size (mean particle size: 358 nm). These DOX-NDs exhibited lower ultrasound activation thresholds (2.46 MPa). By utilizing a single diagnostic transducer for both ultrasound stimulation and imaging guidance, we successfully vaporized the DOX-NDs and released the drug at the tumor site in 4 T1 tumor-bearing mice. Remarkably, the PND group achieved similar tumor remission effects with less than half the dose of DOX required in conventional treatment. Furthermore, the ultrasound-mediated vaporization of DOX-NDs induced tumor cell apoptosis with minimal damage to surrounding normal tissues. In summary, our PND strategy offers a precise and programmable approach for drug delivery and therapy, combining ultrasound imaging guidance. This approach shows great potential in enhancing tumor treatment efficacy while minimizing harm to healthy tissues.

Hypomorphic temperature-sensitive alleles of NSDHL cause CK syndrome.

CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development.

Optimized U-Net model for 3D light-sheet image segmentation of zebrafish trunk vessels.

The growth of zebrafish's vessels can be used as an indicator of the vascular development process and to study the biological mechanisms. The three-dimensional (3D) structures of zebrafish's trunk vessels could be imaged by state-of-art light-sheet fluorescent microscopy with high efficiency. A large amount of data was then produced. Accurate segmentation of these 3D images becomes a new bottleneck for automatic and quantitative analysis. Here, we propose a Multi-scale 3D U-Net model to perform the segmentation of trunk vessels. The segmentation accuracies of 82.3% and 83.0%, as evaluated by the IoU (Intersection over Union) parameter, were achieved for intersegmental vessels and the dorsal longitudinal anastomotic vessels respectively. The growth of zebrafish vasculature from 42-62 hours was then analyzed quantitatively.

Development and Primary Application of an Indirect ELISA Based on Rep Protein to Analyze Antibodies against Porcine Cocirvirus-like Virus (PCLV).

Porcine circovirus-like virus (PCLV) is a member of circovirus that contains a single-strand DNA genome, which may be one of the pathogens that causes diarrheal symptoms in pigs. The Rep protein encoded by the genome of PCLV may be responsible for viral genome replication. The development of serological detection methods for PCLV is of great necessity for clinical diagnosis, as well as epidemiological investigations. Therefore, this study attempted to build an indirect enzyme-linked immunosorbent assay (ELISA) to examine antibodies against PCLV based on the His-tagged recombinant Rep protein. Full-length PCLV Rep protein was induced and expressed in E. coli and was purified as an antigen to establish an ELISA detection kit. The purified Rep protein was used to inject into mice to produce specific antibodies. There was no cross-reaction of Rep-based ELISA with antisera against other porcine viruses. The intra-assay and inter-assay coefficient variations (CVs) were 0.644-8.211% and 0.859-7.246%, respectively, indicating good repeatability. The non-cross-reaction with TGEV, PRRSV and PCV2 testing showed high sensitivity and high specificity for this ELISA assay. A total of 1593 serum samples collected from different pig farms in Jiangxi Province were tested for anti-PCLV Rep antibodies, and 284 (17.83%) of the 1593 samples were Rep antibody positive. Altogether, the indirect ELISA detection tool developed in this study could be applied to examine serum of PCLV antibodies with good repeatability, high sensitivity and high specificity. In addition, field sample detection results suggested that the PCLV antibody has a low prevalence in pig populations in Jiangxi Province of China.

OTUD6A promotes prostate tumorigenesis via deubiquitinating Brg1 and AR.

Ovarian tumor (OTU) subfamily deubiquitinases are involved in various cellular processes, such as inflammation, ferroptosis and tumorigenesis; however, their pathological roles in prostate cancer (PCa) remain largely unexplored. In this study, we observed that several OTU members displayed genomic amplification in PCa, among which ovarian tumor deubiquitinase 6A (OTUD6A) amplified in the top around 15-20%. Further clinical investigation showed that the OTUD6A protein was highly expressed in prostate tumors, and increased OTUD6A expression correlated with a higher biochemical recurrence risk after prostatectomy. Biologically, wild-type but not a catalytically inactive mutant form of OTUD6A was required for PCa cell progression. In vivo experiments demonstrated that OTUD6A oligonucleotides markedly suppressed prostate tumorigenesis in PtenPC-/- mice and patient-derived xenograft (PDX) models. Mechanistically, the SWI/SNF ATPase subunit Brg1 and the nuclear receptor AR (androgen receptor) were identified as essential substrates for OTUD6A in PCa cells by a mass spectrometry (MS) screening approach. Furthermore, OTUD6A stabilized these two proteins by erasing the K27-linked polyubiquitination of Brg1 and K11-linked polyubiquitination of AR. OTUD6A amplification exhibited strong mutual exclusivity with mutations in the tumor suppressors FBXW7 and SPOP. Collectively, our results indicate the therapeutic potential of targeting OTUD6A as a deubiquitinase of Brg1 and AR for PCa treatment.

Ultrasound microvasculature imaging with entropy-based radiality super-resolution (ERSR).

Objective:Microvasculature is highly relevant to the occurrence and development of pathologies such as cancer and diabetes. Ultrasound localization microscopy (ULM) has bypassed the diffraction limit and demonstrated its great potential to provide new imaging modality and establish new diagnostic criteria in clinical application. However, sparse microbubble distribution can be a significant bottleneck for improving temporal resolution, even for further clinical translation. Other important challenges for ULM to tackle in clinic also include high microbubble concentration and low frame rate.Approach:As part of the efforts to facilitate clinical translation, this paper focused on the low frame rate and the high microbubble distribution issue and proposed a new super-resolution imaging strategy called entropy-based radiality super-resolution (ERSR). The feasibility of ERSR is validated with simulations, phantom experiment and contrast-enhanced ultrasound scan of rabbit sciatic nerve with clinical accessible ultrasound system.Main results:ERSR can achieve 10 times improvement in spatial resolution compared to conventional ultrasound imaging, higher temporal resolution (∼10 times higher) and contrast-to-noise ratio under high-density microbubbles, compared with ULM under low-density microbubbles.Significance:We conclude ERSR could be a valuable imaging tool with high spatio-temporal resolution for clinical diagnosis and assessment of diseases potentially.

Blinking Acoustic Nanodroplets Enable Fast Super-resolution Ultrasound Imaging.

The advent of localization-based super-resolution ultrasound (SRUS) imaging creates a vista for precision vasculature and hemodynamic measurements in brain science, cardiovascular diseases, and cancer. As blinking fluorophores are crucial to super-resolution optical imaging, blinking acoustic contrast agents enabling ultrasound localization microscopy have been highly sought, but only with limited success. Here we report on the discovery and characterization of a type of blinking acoustic nanodroplets (BANDs) ideal for SRUS. BANDs of 200-500 nm diameters comprise a perfluorocarbon-filled core and a shell of DSPC, Pluronic F68, and DSPE-PEG2000. When driven by clinically safe acoustic pulses (MI < 1.9) provided by a diagnostic ultrasound transducer, BANDs underwent reversible vaporization and reliquefaction, manifesting as "blinks", at rates of up to 5 kHz. By sparse activation of perfluorohexane-filled BANDs-C6 at high concentrations, only 100 frames of ultrasound imaging were sufficient to reconstruct super-resolution images of a no-flow tube through either cumulative localization or temporal radiality autocorrelation. Furthermore, the use of high-density BANDs-C6-4 (1 × 108/mL) with a 1:9 admixture of perfluorohexane and perfluorobutane supported the fast SRUS imaging of muscle vasculature in live animals, at 64 µm resolution requiring only 100 frames per layer. We anticipate that the BANDs developed here will greatly boost the application of SRUS in both basic science and clinical settings.

Characterization of a new X-linked mental retardation syndrome with microcephaly, cortical malformation, and thin habitus.

X-linked mental retardation (XLMR) affects 1-2/1,000 males and accounts for approximately 10% of all mental retardation (MR). We have ascertained a syndromic form of XLMR segregating within a five-generation family with seven affected males. Prominent characteristics include mild to severe MR, cortical malformation, microcephaly, seizures, thin build with distinct facial features including a long and thin face, epicanthic folds, almond-shaped eyes, upslanting palpebral fissures and micrognathia and behavioral problems. Carrier females have normal physical appearance and intelligence. This combination of features is unreported and distinct from Lujan-Fryns syndrome, Snyder-Robinson syndrome, and zinc finger DHHC domain-containing 9-associated MR. We propose the name of this new syndrome to be CK syndrome.

Pristimerin induces apoptosis of oral squamous cell carcinoma cells via G1 phase arrest and MAPK/Erk1/2 and Akt signaling inhibition.

Pristimerin is an active compound isolated from the traditional Chinese herbs Celastraceae and Hippocrateaceae. It has been reported to exert antitumor effects under experimental and clinical conditions; however, the antitumor effects and underlying mechanisms of pristimerin in oral cancer cells have not yet been identified. In the present study, the anticancer potential of pristimerin was investigated in two oral squamous cell carcinoma (OSCC) cell lines, CAL-27 and SCC-25. Results demonstrated that pristimerin was toxic against the two cell lines, and exhibited inhibitory effects against proliferation. Furthermore, pristimerin exhibited a more potent anti-proliferative activity in CAL-27 and SCC-25 cells than the common chemotherapy drugs cisplatin and 5-fluorouracil. In addition, cell cycle distribution analysis revealed that G0/G1 phase arrest was induced following pristimerin treatment in CAL-27 and SCC-25 cells, which was strongly associated with upregulation of p21 and p27, coupled with downregulation of cyclin D1 and cyclin E. Meanwhile, pristimerin induced significant apoptosis of CAL-27 and SCC-25 cells, alongside decreased levels of caspase-3 and specific cleavage of poly (ADP-ribose) polymerase. These effects were associated with inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways. With regards to these results, pristimerin may be considered a potent novel active substance for the treatment of OSCC.

Overexpression of miR-4433 by suberoylanilide hydroxamic acid suppresses growth of CML cells and induces apoptosis through targeting Bcr-Abl.

Background: Targeting Bcr-Abl is the key for the treatment of CML. Although great progress has been achieved for the treatment of CML patients in chronic stage, effective drugs with good safety are not available for those in advanced stages of CML patients. In present study, a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was used to screen for microRNA that can target Bcr-Abl. Methods: RT-qPCR was used to determine Bcr-Abl and miR-4433 transcription level in CML cells. In CML cells, Proteins including PARP, caspase-3, acetyl-histone 3, histone 3 and Bcr-Abl, as well as Bcr-Abl downstream proteins were detected using western blot. Cell viability and apoptosis were monitored respectively by MTS assay and flow cytometry. The correlation between miR-4433 and Bcr-Abl was determined by luciferase reporter assay. The anti-tumor effect of miR-4433 to K562 cells was evaluated by nude mouse xenograft model in vivo. Results: SAHA up-regulated the acetylation level of histone 3, and effectively inhibited Bcr-Abl mRNA level and its downstream signal transduction pathway, while inhibiting the growth of CML cells and inducing apoptosis. Furthermore, bioinformatics tools predicted that miR-4433 is a putative microRNA targeting Bcr-Abl and that the expression level of miR-4433 was significantly increased after SAHA treatment in K562 cells. Luciferase activity analysis revealed that miR-4433 directly targets Bcr-Abl. Additionally, transient expression of miR-4433 abrogated Bcr-Abl activity and its downstream signaling pathways while inducing apoptosis in K562 cells. Moreover, stable expression of miR-4433 suppressed Bcr-Abl and its downstream signaling pathway, and inhibited the growth of K562 cells in vitro and the growth of K562-xenografts in nude mice. Conclusion: miR-4433 was identified as a microRNA targeting Bcr-Abl, which may be subject to epigenetic regulation of SAHA, a histone deacetylase inhibitor that has been approved by the US FDA for the treatment of cutaneous T-cell lymphoma. The findings of this study provide a molecular basis from another angle for the use of SAHA in the treatment of CML.

[Tumor metastasis in patients with non-small cell lung cancer is inversely correlated with the number of tumor-infiltrating CD8+ T cells].

Objective To analyze the relationship between the number of invasive T cells in in situ tumors and the clinical metastasis and prognosis in patients with non-small cell lung cancer (NSCLC). Methods A total of 140 lung cancer patients (including 43 cases with metastasis) were selected to observe the infiltration state of CD4+ T cells and CD8+ T cells in tumor tissues by immunohistochemical staining. The infiltration of CD4+ T cells and CD8+ T cells were compared between non-metastatic and metastatic patients. The effects of different infiltration levels of CD4+ T cells and CD8+ T cells on clinical prognosis were analyzed. Results The proportion of CD4+ T cells in the tumor tissues of the two groups was not significantly different. The number of CD8+ T cells in the metastatic group was significantly lower than that in the metastatic group, and the ratio of CD8/CD4 in the non-metastasis group was remarkably higher than that in the metastatic group. Patients with high CD8+ T cell infiltration level or high ratio of CD8/CD4 have a significantly better overall survival rate than the patients with low CD8+ T cell infiltration level or low ratio of CD8/CD4. Conclusion The number of CD8+ T cell infiltration and CD8/CD4 ratio in the tumor tissues of patients with metastatic NSCLC are significantly reduced, which is closely related to the poor prognosis of patients with NSCLC metastasis.