Fork PCR: a universal and efficient genome-walking tool

The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes

Future Arctic Climate Change in CMIP6 Strikingly Intensified by NEMO‐Family Climate Models

Climate change in the Arctic has substantial impacts on human life and ecosystems both within and beyond the Arctic. Our analysis of CMIP6 simulations shows that some climate models project much larger Arctic climate change than other models, including changes in sea ice, ocean mixed layer, air-sea heat flux, and surface air temperature in wintertime. In particular, dramatic enhancement of Arctic Ocean convection down to a few hundred meters is projected in these models but not in others. Interestingly, these models employ the same ocean model family (NEMO) while the choice of models for the atmosphere and sea ice varies. The magnitude of Arctic climate change is proportional to the strength of the increase in poleward ocean heat transport, which is considerably higher in this group of models. Establishing the plausibility of this group of models with high Arctic climate sensitivity to anthropogenic forcing is imperative given the implied ramifications

AutoDiffusion: Training-Free Optimization of Time Steps and Architectures for Automated Diffusion Model Acceleration

Diffusion models are emerging expressive generative models, in which a large number of time steps (inference steps) are required for a single image generation. To accelerate such tedious process, reducing steps uniformly is considered as an undisputed principle of diffusion models. We consider that such a uniform assumption is not the optimal solution in practice; i.e., we can find different optimal time steps for different models. Therefore, we propose to search the optimal time steps sequence and compressed model architecture in a unified framework to achieve effective image generation for diffusion models without any further training. Specifically, we first design a unified search space that consists of all possible time steps and various architectures. Then, a two stage evolutionary algorithm is introduced to find the optimal solution in the designed search space. To further accelerate the search process, we employ FID score between generated and real samples to estimate the performance of the sampled examples. As a result, the proposed method is (i).training-free, obtaining the optimal time steps and model architecture without any training process; (ii). orthogonal to most advanced diffusion samplers and can be integrated to gain better sample quality. (iii). generalized, where the searched time steps and architectures can be directly applied on different diffusion models with the same guidance scale. Experimental results show that our method achieves excellent performance by using only a few time steps, e.g. 17.86 FID score on ImageNet 64 $\times$ 64 with only four steps, compared to 138.66 with DDIM. The code is available at https://github.com/lilijiangg/AutoDiffusion

Practice of pharmaceutical services and prescription analysis in internet-based psychiatric hospitals during COVID-19 pandemic in Wuxi, China

ObjectiveTo study the practice of pharmaceutical services in internet-based psychiatric hospitals, and to analyze the prescriptions to ensure the safety and efficacy of internet-based medication in Wuxi, China.MethodsAll 1,259 internet-based prescriptions from our hospital in 2022 were collected, and data on patients’ age, gender, diagnosis, medications used, medication types, dosage forms, rationality of medication use, and reasons for irrationality were analyzed through descriptive statistics.ResultsIn the electronic prescriptions of internet-based psychiatric hospitals, females accounted for the majority (64.50%), with a female-to-male ratio of 1.82:1. Middle-aged and young adults accounted for the majority of patients (57.50%). There were 47 diagnosed diseases involved, with 89 types of medications used and 1,938prescriptions issued. Among them, there were 78 types of western medicine with 1,876 prescriptions (96.80%), and 11 types of traditional Chinese medicine with 62 prescriptions (3.20%). The main medications used were anti-anxiety and antidepressant medications (44.94%) and psychiatric medications (42.21%). The dosage forms were all oral, with tablets (78.53%), capsules (17.54%), and solution preparations (2.17%) being the top three in frequency. According to the prescription review results, the initial pass rate of internet-based system review was 64.26%. After intervention by the internet-based system and manual review by pharmacist reviewers, the final pass rate of internet-based prescriptions reached 99.76%.ConclusionThe practice of pharmaceutical services and prescription analysis in internet-based psychiatric hospitals could significantly improve medication rationality, which fills the research gap in this field. In addition, it promotes the transformation of pharmaceutical service models

How early can myocardial iron overload occur in Beta thalassemia major?

BACKGROUND: Myocardial siderosis is the most common cause of death in patients with beta thalassemia major(TM). This study aimed at investigating the occurrence, prevalence and severity of cardiac iron overload in a young Chinese population with beta TM. METHODS AND RESULTS: We analyzed T2* cardiac magnetic resonance (CMR), left ventricular ejection fraction (LVEF) and serum ferritin (SF) in 201 beta TM patients. The median age was 9 years old. Patients received an average of 13 units of blood per year. The median SF level was 4536 ng/ml and 165 patients (82.1%) had SF>2500 ng/ml. Myocardial iron overload was detected in 68 patients (33.8%) and severe myocardial iron overload was detected in 26 patients (12.6%). Twenty-two patients ≤10 years old had myocardial iron overload, three of whom were only 6 years old. No myocardial iron overload was detected under the age of 6 years. Median LVEF was 64% (measured by CMR in 175 patients). Five of 6 patients with a LVEF<56% and 8 of 10 patients with cardiac disease had myocardial iron overload. CONCLUSIONS: The TM patients under follow-up at this regional centre in China patients are younger than other reported cohorts, more poorly-chelated, and have a high burden of iron overload. Myocardial siderosis occurred in patients younger than previously reported, and was strongly associated with impaired LVEF and cardiac disease. For such poorly-chelated TM patients, our data shows that the first assessment of cardiac T2* should be performed as early as 6 years old

Screening and Stability Evaluation of Angiotensin Converting Enzyme Inhibitory Peptides from Bangia fusco-purpurea

In this study, peptide fractions (F1-F4) with different molecular masses were obtained from Bangia fusco-purpurea through enzymatic hydrolysis and ultrafiltration. F2, with molecular masses of 800–2 000 Da, exhibited the highest in vitro angiotensin-converting enzyme (ACE) inhibitory activity as determined by high performance liquid chromatography (HPLC). The amino acid sequence of F2 was identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and de novo sequencing using PEAKS Studio software. Six ACE inhibitory peptides that stably bind to ACE were selected through molecular docking. The predicted peptides were synthesized by solid-phase synthesis and their in vitro ACE inhibitory activity was verified. Among them, L1 (LVLLFLFGE) showed the highest ACE inhibitory activity with a half maximal inhibitory concentration (IC50) value of 14.22 μg/mL. Molecular docking results indicated that the inhibition of ACE by L1 was mainly attributed to its ability to form hydrogen bond interactions with the active site of ACE. Finally, the effects of temperature, pH, metal ions, light exposure, and simulated gastrointestinal digestion on the stability of L1 were investigated. The results revealed that L1 was highly stable to heat and ionic strength. However, its activity gradually decreased at pH > 2, and was affected by ultraviolet treatment. The ACE inhibitory activity of L1 decreased after simulated gastric and intestinal digestion, but was still significant

Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components

NLRs (nucleotide-binding domain leucine-rich repeat proteins or NOD-like receptors) are regulators of inflammation and immunity. A subgroup of NLRs and the innate immune receptor, AIM2 (absent-in-melanoma 2), can induce the assembly of a large caspase-1 activating complex called the inflammasome. Other NLRs regulate key signaling pathways such as NF-kB and MAPK. Since inflammation is a central component of colorectal cancer (CRC), this work was undertaken to analyze NLR and AIM2 expression in human CRC by combining bioinformatics analysis and experimental verification using clinical tissue samples. Additional experiments analyzed the association of (i) gene expression and cancer staging, and (ii) gene expression among inflammasome components

The dual role of glioma exosomal microRNAs: glioma eliminates tumor suppressor miR-1298-5p via exosomes to promote immunosuppressive effects of MDSCs

Clear evidence shows that tumors could secrete microRNAs (miRNAs) via exosomes to modulate the tumor microenvironment (TME). However, the mechanisms sorting specific miRNAs into exosomes are still unclear. In order to study the biological function and characterization of exosomal miRNAs, we performed whole-transcriptome sequencing in 59 patients’ whole-course cerebrospinal fluid (CSF) small extracellular vesicles (sEV) and matched glioma tissue samples. The results demonstrate that miRNAs could be divided into exosome-enriched miRNAs (ExomiRNAs) and intracellular-retained miRNAs (CLmiRNAs), and exosome-enriched miRNAs generally play a dual role. Among them, miR-1298-5p was enriched in CSF exosomes and suppressed glioma progression in vitro and vivo experiments. Interestingly, exosomal miR-1298-5p could promote the immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) to facilitate glioma. Therefore, we found miR-1298-5p had different effects on glioma cells and MDSCs. Mechanically, downstream signaling pathway analyses showed that miR-1298-5p plays distinct roles in glioma cells and MDSCs via targeting SETD7 and MSH2, respectively. Moreover, reverse verification was performed on the intracellular-retained miRNA miR-9-5p. Thus, we confirmed that tumor-suppressive miRNAs in glioma cells could be eliminated through exosomes and target tumor-associated immune cells to induce tumor-promoting phenotypes. Glioma could get double benefit from it. These findings uncover the mechanisms that glioma selectively sorts miRNAs into exosomes and modulates tumor immunity.publishedVersio

SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner

As one of the most common post-transcriptional modifications of mRNAs and noncoding RNAs, N6-methyladenosine (m6A) modification regulates almost every aspect of RNA metabolism. Evidence indicates that dysregulation of m6A modification and associated proteins contributes to glioblastoma (GBM) progression. However, the function of fat mass and obesity-associated protein (FTO), an m6A demethylase, has not been systematically and comprehensively explored in GBM. Here, we found that decreased FTO expression in clinical specimens correlated with higher glioma grades and poorer clinical outcomes. Functionally, FTO inhibited growth and invasion in GBM cells in vitro and in vivo. Mechanistically, FTO regulated the m6A modification of primary microRNA-10a (pri-miR-10a), which could be recognized by reader HNRNPA2B1, recruiting the microRNA microprocessor complex protein DGCR8 and mediating pri-miR-10a processing. Furthermore, the transcriptional activity of FTO was inhibited by the transcription factor SPI1, which could be specifically disrupted by the SPI1 inhibitor DB2313. Treatment with this inhibitor restored endogenous FTO expression and decreased GBM tumor burden, suggesting that FTO may serve as a novel prognostic indicator and therapeutic molecular target of GBM.publishedVersio

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits