Simple and efficient expression of codon-optimized mouse leukemia inhibitory factor in Escherichia coli

Purpose: To obtain a higher yield of mouse leukemia inhibitory factor to maintain the proliferation potential of pluripotent stem cells at a low cost.Methods: A method was designed to produce recombinant mLIF protein (rmLIF) in Escherichia coli. Through analysis of rmLIF sequence, it was found that rare codons were interspersed. After mutation from rare codons to Escherichia coli (E. coli) preferred ones were selected, the mutated gene mLIFm was cloned into pET15b vector. The pET15b-mLIFm was then transformed into Rosetta-gami strain and induced with optimal conditions at 18 oC for 16 h. Mass spectrometry was carried out to identify the peptides.Results: After purification, the yield of the codon-optimized rmLIFm was 141 mg/L, compared with 110 mg/L for the original rmLIF. Mass spectral analysis showed the presence of four major peptides each with an intensity > 10 % at m/z 1031.57, 1539.82, 1412.01 and 2229.10 in mLIFm, respectively. Histagged rmLIFm fusion protein displayed the potential to maintain the morphology of undifferentiated mouse embryonic stem cells (mESCs), which were positive for mESCs markers (Oct-4, Nanog, Sox-2, stage-specific embryonic antigen-1).Conclusion: The findings provide a means to produce mLIF in a short, useful, cost-effective and environmentally-friendly manner, and thus lays a foundation for further studies of mLIF.Keywords: Leukemia inhibitory factor, Mutated gene, Protein expression, Purification, Stem cells, Peptides, Escherichia col

An integrated genetic linkage map for silkworms with three parental combinations and its application to the mapping of single genes and QTL

<p>Abstract</p> <p>Background</p> <p><it>Bombyx mori</it>, the domesticated silkworm, is a well-studied model insect with great economic and scientific significance. Although more than 400 mutations have been described in silkworms, most have not been identified, especially those affecting economically-important traits. Simple sequence repeats (SSRs) are effective and economical tools for mapping traits and genetic improvement. The current SSR linkage map is of low density and contains few polymorphisms. The purpose of this work was to develop a dense and informative linkage map that would assist in the preliminary mapping and dissection of quantitative trait loci (QTL) in a variety of silkworm strains.</p> <p>Results</p> <p>Through an analysis of > 50,000 genotypes across new mapping populations, we constructed two new linkage maps covering 27 assigned chromosomes and merged the data with previously reported data sets. The integrated consensus map contains 692 unique SSR sites, improving the density from 6.3 cM in the previous map to 4.8 cM. We also developed 497 confirmed neighboring markers for corresponding low-polymorphism sites, with 244 having polymorphisms. Large-scale statistics on the SSR type were suggestive of highly efficient markers, based upon which we searched 16,462 available genomic scaffolds for SSR loci. With the newly constructed map, we mapped single-gene traits, the QTL of filaments, and a number of ribosomal protein genes.</p> <p>Conclusion</p> <p>The integrated map produced in this study is a highly efficient genetic tool for the high-throughput mapping of single genes and QTL. Compared to previous maps, the current map offers a greater number of markers and polymorphisms; thus, it may be used as a resource for marker-assisted breeding.</p

Clinical Findings in Patients With Persistent Positional Nystagmus: The Designation of “Heavy and Light Cupula”

Objective: Direction-changing positional nystagmus (DCPN) had been observed as persistent horizontal apogeotropic and was considered as “cupulolithiasis or heavy cupula. ” Recently, the concept of “light cupula” exhibiting persistent geotropic DCPN has been introduced. However, the light cupula is not systematically described, while the identification and diagnosis of “light cupula” should be improved. Here we investigated the underlying characteristics and therapeutic options designed to the “light” and “heavy” cupula, respectively; and summarized the clinical characteristics and therapeutic effect in the two groups.Methods: A total of 359 cases with vertigo and bilateral DCPN were found in the supine roll test. Only 25 patients with persistent DCPN were enrolled and followed up. According to the direction of nystagmus, we further divided the patients into “heavy cupula” (apogeotropic) and “light cupula” (geotropic) groups. We compared the incidence, characteristics of nystagmus and the efficacy of repositioning maneuver in the two groups.Results: Nine patients with persistent horizontal geotropic DCPN were confirmed as “light cupula,” other 16 patients with persistent horizontal ageotropic DCPN were confirmed as heavy cupula. All 25 patients had null plane; the mean value and standard deviation of the null plane in light cupula and heavy cupula was 25.67 ± 9.31° and 27.06 ± 6.29°, respectively. The mean value and standard deviation of the termination plane in light cupula was 28.78 ± 10.00°, and 30.25 ± 6.53° in heavy cupula. There was no statistical significance between the two groups. We found that the direction of evoked nystagmus in the supine position was toward the intact side in light cupula, while in heavy cupula, it was toward the lesion side. The null plane appeared on the lesion side. For light cupula patients, the effect was not obvious at Day-7 after the treatment, however, treatment for most heavy cupula patients were effective. All patients recovered after 30 days of treatment.Conclusion: The null plane is crucial in determining the lesion side for light or heavy cupula. Although the short-term therapeutic effect of the light cupula is not as promising as the effect seen in heavy cupula, the long-term prognosis in both groups is comparable; with all patients recovered after 30 days of treatment.Study design: This is a retrospective cohort study

Panel 3 : Genetics and Precision Medicine of Otitis Media

Objective. The objective is to perform a comprehensive review of the literature up to 2015 on the genetics and precision medicine relevant to otitis media. Data Sources. PubMed database of the National Library of Medicine. Review Methods. Two subpanels were formed comprising experts in the genetics and precision medicine of otitis media. Each of the panels reviewed the literature in their respective fields and wrote draft reviews. The reviews were shared with all panel members, and a merged draft was created. The entire panel met at the 18th International Symposium on Recent Advances in Otitis Media in June 2015 and discussed the review and refined the content. A final draft was made, circulated, and approved by the panel members. Conclusion. Many genes relevant to otitis media have been identified in the last 4 years in advancing our knowledge regarding the predisposition of the middle ear mucosa to commensals and pathogens. Advances include mutant animal models and clinical studies. Many signaling pathways are involved in the predisposition of otitis media. Implications for Practice. New knowledge on the genetic background relevant to otitis media forms a basis of novel potential interventions, including potential new ways to treat otitis media.Peer reviewe

Reconstruction of global gridded monthly sectoral water withdrawals for 1971-2010 and analysis of their spatiotemporal patterns

Human water withdrawal has increasingly altered the global water cycle in past decades, yet our understanding of its driving forces and patterns is limited. Reported historical estimates of sectoral water withdrawals are often sparse and incomplete, mainly restricted to water withdrawal estimates available at annual and country scale, due to a lack of observations at local and seasonal time scales. In this study, through collecting and consolidating various sources of reported data and developing spatial and temporal statistical downscaling algorithms, we reconstruct a global monthly gridded (0.5 degree) sectoral water withdrawal dataset for the period 1971–2010, which distinguishes six water use sectors, i.e. irrigation, domestic, electricity generation (cooling of thermal power plants), livestock, mining, and manufacturing. Based on the reconstructed dataset, the spatial and temporal patterns of historical water withdrawal are analyzed. Results show that global total water withdrawal has increased significantly during 1971–2010, mainly driven by the increase of irrigation water withdrawal. Regions with high water withdrawal are those densely populated or with large irrigated cropland production, e.g., the United States (US), eastern China, India, and Europe. Seasonally, irrigation water withdrawal in summer for the major crops contributes a large percentage of annual total irrigation water withdrawal in mid and high-latitude regions, and the dominant season of irrigation water withdrawal is also different across regions. Domestic water withdrawal is mostly characterized by a summer peak, while water withdrawal for electricity generation has a winter peak in high-latitude regions and a summer peak in low-latitude regions. Despite the overall increasing trend, irrigation in the western US and domestic water withdrawal in western Europe exhibit a decreasing trend. Our results highlight the distinct spatial pattern of human water use by sectors at the seasonal and annual scales. The reconstructed gridded water withdrawal dataset is open-access, and can be used for examining issues related to water withdrawals at fine spatial, temporal and sectoral scales

Correlation analysis between foot deformity and diabetic foot with radiographic measurement

BackgroundFoot deformity is a risk factor for diabetic foot ulcer. This study was aimed to investigate the relationship between hallux valgus (HV) and diabetic foot through the radiographic measurement.MethodsThe patients with diabetic foot hospitalizing in the Department of Endocrinology, the First Affiliated Hospital of Chongqing Medical University from September 2016 to June 2020 were selected. Then the foot plain X-ray radiographs were completed, and the size of HV angle (HVA) was measured. Their clinical data were collected, and the ulcer recurrence rate, amputation rate and mortality rate of the patients were followed up.ResultsA total of 370 patients were included. According to HVA, patients were divided into non-HV group (HVA&lt;15°), and mild (15°≤HVA ≤ 20°), moderate (20°&lt;HVA ≤ 40°) and severe (HVA&gt;40°) HV groups. The age, height, BMI, smoking history and glycosylated hemoglobin level among the non-HVA, mild, moderate, and severe HV group (P&lt;0.05), while smoking history, HbA1c, eGFR and autonomic neuropathy were significantly lower in HV group than those in non-HV group (P&lt;0.05). The ulcer area in patients with moderate HV was larger than that in non-HV patients, and the severity of infection in patients with severe HV was significantly higher than that the other three groups (P&lt;0.05).ConclusionThe occurrence of HV is not only related to age and BMI, but also to the creatinine and eGFR level, autonomic neuropathy, lower limb arteriosclerosis occlusion, coronary heart disease and hypertension. Therefore, more attention should be paid to renal function screening, neuropathy screening and evaluation of lower extremity vascular lesions in patients with diabetes, especially those with moderate or higher HV

Single-cell transcriptomics of hepatic stellate cells uncover crucial pathways and key regulators involved in non-alcoholic steatohepatitis

Background: Fibrosis is an important pathological process in the development of non-alcoholic steatohepatitis (NASH), and the activation of hepatic stellate cell (HSC) is a central event in liver fibrosis. However, the transcriptomic change of activated HSCs (aHSCs) and resting HSCs (rHSCs) in NASH patients has not been assessed. This study aimed to identify transcriptomic signature of HSCs during the development of NASH and the underlying key functional pathways. Methods: NASH-associated transcriptomic change of HSCs was defined by single-cell RNA-sequencing (scRNA-seq) analysis, and those top upregulated genes were identified as NASH-associated transcriptomic signatures. Those functional pathways involved in the NASH-associated transcriptomic change of aHSCs were explored by weighted gene co-expression network analysis (WGCNA) and functional enrichment analyses. Key regulators were explored by upstream regulator analysis and transcription factor enrichment analysis. Results: scRNA-seq analysis identified numerous differentially expressed genes in both rHSCs and aHSCs between NASH patients and healthy controls. Both scRNA-seq analysis and in-vivo experiments showed the existence of rHSCs (mainly expressing a-SMA) in the normal liver and the increased aHSCs (mainly expressing collagen 1) in the fibrosis liver tissues. NASH-associated transcriptomic signature of rHSC (NASHrHSCsignature) and NASH-associated transcriptomic signature of aHSC (NASHaHSCsignature) were identified. WGCNA revealed the main pathways correlated with the transcriptomic change of aHSCs. Several key upstream regulators and transcription factors for determining the functional change of aHSCs in NASH were identified. Conclusion: This study developed a useful transcriptomic signature with the potential in assessing fibrosis severity in the development of NASH. This study also identified the main pathways in the activation of HSCs during the development of NASH

Dissecting causal associations of type 2 diabetes with 111 types of ocular conditions: a Mendelian randomization study

BackgroundDespite the well-established findings of a higher incidence of retina-related eye diseases in patients with diabetes, there is less investigation into the causal relationship between diabetes and non-retinal eye conditions, such as age-related cataracts and glaucoma.MethodsWe performed Mendelian randomization (MR) analysis to examine the causal relationship between type 2 diabetes mellitus (T2DM) and 111 ocular diseases. We employed a set of 184 single nucleotide polymorphisms (SNPs) that reached genome-wide significance as instrumental variables (IVs). The primary analysis utilized the inverse variance-weighted (IVW) method, with MR-Egger and weighted median (WM) methods serving as supplementary analyses.ResultsThe results revealed suggestive positive causal relationships between T2DM and various ocular conditions, including “Senile cataract” (OR= 1.07; 95% CI: 1.03, 1.11; P=7.77×10-4), “Glaucoma” (OR= 1.08; 95% CI: 1.02, 1.13; P=4.81×10-3), and “Disorders of optic nerve and visual pathways” (OR= 1.10; 95% CI: 0.99, 1.23; P=7.01×10-2).ConclusionOur evidence supports a causal relationship between T2DM and specific ocular disorders. This provides a basis for further research on the importance of T2DM management and prevention strategies in maintaining ocular health