The end-of-life power battery recycling & remanufacturing center location-adjustment problem considering battery capacity and quantity uncertainty.

The booming electric vehicle market has led to an increasing number of end-of-life power batteries. In order to reduce environmental pollution and promote the realization of circular economy, how to fully and effectively recycle the end-of-life power batteries has become an urgent challenge to be solved today. The recycling & remanufacturing center is an extremely important and key facility in the recycling process of used batteries, which ensures that the recycled batteries can be handled in a standardized manner under the conditions of professional facilities. In reality, different adjustment options for existing recycling & remanufacturing centers have a huge impact on the planning of new sites. This paper proposes a mixed-integer linear programming model for the siting problem of battery recycling & remanufacturing centers considering site location-adjustment. The model allows for demolition, renewal, and new construction options in planning for recycling & remanufacturing centers. By adjusting existing sites, this paper provides an efficient allocation of resources under the condition of meeting the demand for recycling of used batteries. Next, under the new model proposed in this paper, the uncertainty of the quantity and capacity of recycled used batteries is considered. By establishing different capacity conditions of batteries under multiple scenarios, a robust model was developed to determine the number and location of recycling & remanufacturing centers, which promotes sustainable development, reduces environmental pollution and effectively copes with the risk of the future quantity of used batteries exceeding expectations. In the final results of the case analysis, our proposed model considering the existing sites adjustment reduces the cost by 3.14% compared to the traditional model, and the average site utilization rate is 15.38% higher than the traditional model. The results show that the model has an effective effect in reducing costs, allocating resources, and improving efficiency, which could provide important support for decision-making in the recycling of used power batteries.

The occurrence of hypogonadotropic hypogonadism in Chinese men with type 2 diabetes.

CONTEXT: The previous studies showed that hypogonadotropic hypogonadism (HH) occurred commonly in men with type 2 diabetes. However, since all the cohorts tested were from American and European studies, the occurrence of HH/nongonadal illness (NGI) in Chinese populations is unclear. OBJECTIVE: The study aimed to explore the occurrence of HH/NGI in Chinese men with type 2 diabetes. Furthermore, the correlative factors and predictors of hypogonadism were investigated. DESIGN: We conducted a cross-sectional study of 637 Chinese men with type 2 diabetes aged 20-75 years in our clinic. The prevalence of HH/NGI was investigated by measuring serum total testosterone (TT), sex hormone-binding globulin (SHBG), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the enrolled subjects. Free testosterone (FT) was calculated by using SHBG and TT levels and hypogonadism was defined as TT lower than 10.4 nmol/L and calculated FT (cFT) lower than 0.225 nmol/L. The LH cut-off value for defining HH/NGI was 9.4 mIU/ml. RESULTS: The results suggested that 31.9% of male Chinese type 2 diabetes patients had hypogonadism and 26.5% of subjects in our cohort were determined as HH/NGI. The occurrence of hypogonadism was markedly correlated with body mass index (BMI). There was a significant association between TT, cFT and SHBG levels with BMI. TT levels are inversely correlated with BMI and homeostasis model assessment-estimated insulin resistance (HOMA-IR) while positively related with SHBG. The cFT levels were inversely correlated with age, LH, FSH, BMI and HOMA-IR. Multiple regression analysis suggested that SHBG, BMI and HOMA-IR were significant predictors of TT and cFT. CONCLUSION: Our present study offered the first evidence that the occurrence of HH/NGI in Chinese male type 2 diabetes was 26.5%. TT and cFT were significantly correlated with BMI, SHBG and HOMA-IR in Chinese men with type 2 diabetes.

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Prediction of the Potential Mechanism of Triptolide in Improving Diabetic Nephropathy by Utilizing A Network Pharmacology and Molecular Docking Approach.

BACKGROUND: Triptolide (TP) is a major active component of colquhounia root tablet, which has been long been used in China to treat diabetic nephropathy (DN) due to its marked anti­inflammatory, antiproteinuric, and podocyte­protective effects. METHODS: This study investigated the anti-proteinuria activity and related signaling cascade of TP in DN by utilizing a network pharmacology and molecular docking approach. RESULTS: From the GeneCard, DisGeNET, and National Center for Biotechnology Information Gene databases, 1458 DN targets were obtained and input together with 303 TP targets into Venny2.1.0 for mapping and comparing. In total, 113 common targets of TP and DN were obtained, of which 7 targets were found to play an important role through theoretical inhibitory constant analysis. The common targets were further analyzed by Kyoto Encyclopedia of Genes and Genomes to identify the pathways related to the therapeutic effect of TP on DN. Among them, the seven targets were found to play key roles in six signaling pathways. The molecular docking results also showed TP had good binding ability to the seven targets. CONCLUSIONS: Analysis of the common targets and key pathways showed that TP can improve DN via its anti-nephritis, anti-renal fibrosis, antioxidant, and podocyte-protective effects, which might elucidate the mechanism by which TP improves renal function and reduces proteinuria in DN.

Elevated serum uric acid is not an independent risk factor for the occurrence of Type 2 diabetic kidney disease in Chinese populations.

Previous studies suggested that increased serum uric acid (SUA) level is an independent risk factor for albuminuria in Type 2 diabetes (T2D) patients. However, the association between SUA and onset of Type 2 DKD (T2DKD) remained to be clarified. This was a cross-sectional clinical study in which 1210 Chinese T2D patients were enrolled. According to the urine albumin-to-creatinine ratio (UACR), the cohort was divided into normal-albuminuria (UACR < 30 mg/g), micro-albuminuria (UACR 30-300 mg/g) and macro-albuminuria (UACR > 300 mg/g). The micro- and macro-albuminuria groups were combined into albuminuria category. Results showed that T2D patients with macro-albuminuria have significantly higher SUA than the other 2 groups (P < .001). In the binary logistic regression model, the subjects with SUA higher than 420 µmol/L were associated with a 2-fold increase in the odds of albuminuria (odds ratio = 2.024, 95% confidence interval: 1.232-3.325, P = .005), as compared with those with SUA lower than 300 µmol/L. Moreover, the multinomial regression analysis revealed that the subjects with SUA higher than 420 µmol/L had about 3-fold increase in the odds of macro-albuminuria (odds ratio = 3.758, 95% confidence interval: 2.051-6.885, P < .001), as compared with those with SUA lower than 300 µmol/L. However, SUA was not significantly associated with the presence of micro-albuminuria. Although the SUAwas not independently risk factor for micro-albuminuria, it was closely correlated with the development of macro-albuminuria in Chinese T2DKD patients. Elevated SUA may be useful for predicting the occurrence of macro-albuminuria but not onset of micro-albuminuria at the early stage of T2DKD.

PASTMUS: mapping functional elements at single amino acid resolution in human cells.

Identification of functional elements for a protein of interest is important for achieving a mechanistic understanding. However, it remains cumbersome to assess each and every amino acid of a given protein in relevance to its functional significance. Here, we report a strategy, PArsing fragmented DNA Sequences from CRISPR Tiling MUtagenesis Screening (PASTMUS), which provides a streamlined workflow and a bioinformatics pipeline to identify critical amino acids of proteins in their native biological contexts. Using this approach, we map six proteins-three bacterial toxin receptors and three cancer drug targets, and acquire their corresponding functional maps at amino acid resolution.

A surrogate reporter system for multiplexable evaluation of CRISPR/Cas9 in targeted mutagenesis.

Engineered nucleases in genome editing manifest diverse efficiencies at different targeted loci. There is therefore a constant need to evaluate the mutation rates at given loci. T7 endonuclease 1 (T7E1) and Surveyor mismatch cleavage assays are the most widely used methods, but they are labour and time consuming, especially when one must address multiple samples in parallel. Here, we report a surrogate system, called UDAR (Universal Donor As Reporter), to evaluate the efficiency of CRISPR/Cas9 in targeted mutagenesis. Based on the non-homologous end-joining (NHEJ)-mediated knock-in strategy, the UDAR-based assay allows us to rapidly evaluate the targeting efficiencies of sgRNAs. With one-step transfection and fluorescence-activated cell sorting (FACS) analysis, the UDAR assay can be completed on a large scale within three days. For detecting mutations generated by the CRISPR/Cas9 system, a significant positive correlation was observed between the results from the UDAR and T7E1 assays. Consistently, the UDAR assay could quantitatively assess bleomycin- or ICRF193-induced double-strand breaks (DSBs), which suggests that this novel strategy is broadly applicable to assessing the DSB-inducing capability of various agents. With the increasing impact of genome editing in biomedical studies, the UDAR method can significantly benefit the evaluation of targeted mutagenesis, especially for high-throughput purposes.

Genome-Wide CRISPR/Cas9 Screening for High-Throughput Functional Genomics in Human Cells.

It is highly desirable to identify gene's function in a high-throughput fashion, and the CRISPR/Cas9 system has been harnessed to meet such a need. Here, we describe a general method to generate genome-scale lentiviral single-guide RNA (sgRNA) library and conduct a pooled function-based screening in human cells. This protocol would be of interest to researchers to rapidly identify genes in a variety of biological processes.

Simultaneous generation of multi-gene knockouts in human cells.

Genome-editing techniques enable the generation of gene knockouts in various mammalian cell lines. However, it remains technically challenging to completely disrupt a targeted gene using a canonical method in a timely manner. To improve the efficiency of producing reliable genomic modifications, we designed a method using a linear donor fragment containing a reporter system. Combined with a homologous recombination-independent knock-in strategy, we successfully enriched those cell clones that specifically carry the target gene mutations. We observed a much improved success rate when generating single- and multiple-gene knockouts in a one-step procedure using this special protocol coupled with the CRISPR/Cas9 system. This new approach further empowers the molecular biological study of genes and their functions.

High-throughput screens in mammalian cells using the CRISPR-Cas9 system.

As a powerful genome-editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)-clustered regularly interspaced short palindromic repeats-associated protein 9 (Cas9) system has been quickly developed into a large-scale function-based screening strategy in mammalian cells. This new type of genetic library is constructed through the lentiviral delivery of single-guide RNA collections that direct Cas9 or inactive dead Cas9 fused with effectors to interrogate gene function or regulate gene transcription in targeted cells. Compared with RNA interference screening, the CRISPR-Cas9 system demonstrates much higher levels of effectiveness and reliability with respect to both loss-of-function and gain-of-function screening. Unlike the RNA interference strategy, a CRISPR-Cas9 library can target both protein-coding sequences and regulatory elements, including promoters, enhancers and elements transcribing microRNAs and long noncoding RNAs. This powerful genetic tool will undoubtedly accelerate the mechanistic discovery of various biological processes. In this mini review, we summarize the general procedure of CRISPR-Cas9 library mediated functional screening, system optimization strategies and applications of this new genetic toolkit.

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B.

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.