GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.Podocyte dysfunction, injury or loss is a common and decisive cause of various glomerular diseases and understanding the molecular mechanism underlying podocyte response to stress will be very helpful to undermine the pathogenesis of podocyte injury and the targeted therapy for glomerular diseases.The members of Gadd45 gene family, Gadd45a, Gadd45b and Gadd45r have been commonly implicated in stress signaling in response to physiological or environmental stressors, resulting in cell cycle arrest, DNA damage repair, cell survival, senescence and apoptosis.¹ Recently, this gene family has been found differentially expressed in several podocyte injury models. Zhang et al.² observed an induction of GADD45β mRNA expression by lipopolysaccharide in the lung, kidney and spleen, which had the highest GADD45β mRNA expression among all of the tissues examined. Jeffrey W Pippin reported that protein expression of GADD45 was increased in glomeruli from passive Heymann nephritis rats and cultured podocytes exposed in vitro to C5b-9. ³ More recently, Shi et al.⁴ reported that Gadd45b was upregulated in glomeruli of mice with podocyte-specific deletion of Dicer, suggesting the involvement of Gadd45b in podocyte injury. However, no functional characterization of Gadd45 genes in podocytes has been conducted to date and the role of GADD45B in the context of podocyte injury remains unclear.Zebrafish has emerged as a new vertebrate model system for renal glomerular research. The podocytes and renal glomeruli in zebrafish kidney are structurally, molecularly and functionally conserved, rendering zebrafish a valuable and relevant model for podocyte studies. To characterize the role of GADD45b in podocyte injury, we therefore employed zebrafish as an in vivo model system and human podocytes as an in vitro model. We observed the upregulation of GADD45B on podocyte injury in zebrafish renal glomeruli as well as in cultured human podocytes treated with TGF-β and PAN. We further showed that podocyte-specific overexpression of zebrafish orthologs of gadd45b predisposed podocytes to injury, whereas inhibition of gadd45b expression in zebrafish larvae ameliorated podocyte injury and reduced proteinuria. Furthermore, we found that the ROS-GADD45B-p38 pathway was involved in the regulation of GADD45B expression and deleterious role in podocyte injury. Collectively, we have identified GADD45B as an important player in podocyte injury.     

Difference in 24-Hour Urine Composition between Diabetic and Non-Diabetic Adults without Nephrolithiasis

Background

Diabetic patients are more likely to develop kidney stones than the general population. The underlying mechanisms for this disparity remain to be elucidated. Little is known about the relationship between urine composition and diabetes mellitus in non-stone-forming individuals. We sought to examine the differences in the 24-hour (24-h) urine composition between diabetic and non-diabetic adults who were not stone formers.

Methods

A convenience sample of 538 individuals without a history of nephrolithiasis, gout, hyperparathyroidism, or gastroenteric diseases participated in this study. The 24-h urine profiles of 115 diabetic adults were compared with those of 423 non-diabetic adults. Diabetes was defined by self-reported physician diagnosis or medication use. All participants were non-stone formers confirmed by urinary tract ultrasonography. Participants provided a fasting blood sample and a single 24-h urine collection for stone risk analysis. Student’s t-test was used to compare mean urinary values. Linear regression models were adjusted for age, gender, body mass index, hypertension, fasting serum glucose, serum total cholesterol, estimated creatinine clearance rate and urinary factors.

Results

Univariable analysis showed that the diabetic participants had significantly higher 24-h urine volumes and lower urine calcium and magnesium excretions than non-diabetic participants (all P < 0.05). After multivariate adjustment, no significant differences in 24-h urine composition were observed between diabetic and non-diabetic participants except for a slightly increased 24-h urine volume in diabetic participants (all P > 0.05). The main limitation of this study is that the convenience samples and self-reported data may have been sources of bias.

Conclusion

Our data showed that there were no differences in 24-h urine composition between diabetic and non-diabetic adults who are not stone formers. The reason for it might be the improved glycemic control in diabetic individuals in our study. Therefore, a tighter glycemic control might reduce stone formation in diabetic adults.     

Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.     

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Nivolumab, an anti-programmed death (PD)1 IgG₄ antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG₁), panitumumab (IgG₂) and atezolizumab (aglyco-IgG₁) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG₁?1?2?4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG₄ backbone.     

Whole-Genome Sequencing for the Investigation of a Hospital Outbreak of MRSA in China

Staphylococcus aureus is a globally disseminated drug-resistant bacterial species. It remains a leading cause of hospital-acquired infection, primarily among immunocompromised patients. In 2012, the Affiliated People’s Hospital of Jiangsu University experienced a putative outbreak of methicillin-resistant S. aureus (MRSA) that affected 12 patients in the Neurosurgery Department. In this study, whole-genome sequencing (WGS) was used to gain insight into the epidemiology of the outbreak caused by MRSA, and traditional bacterial genotyping approaches were also applied to provide supportive evidence for WGS. We sequenced the DNA from 6 isolates associated with the outbreak. Phylogenetic analysis was constructed by comparing single-nucleotide polymorphisms (SNPs) in the core genome of 6 isolates in the present study and another 3 referenced isolates from GenBank. Of the 6 MRSA sequences in the current study, 5 belonged to the same group, clustering with T0131, while the other one clustered closely with TW20. All of the isolates were identified as ST239-SCCmecIII clones. Whole-genome analysis revealed that four of the outbreak isolates were more tightly clustered into a group and SA13002 together with SA13009 were distinct from the outbreak strains, which were considered non-outbreak strains. Based on the sequencing results, the antibiotic-resistance gene status (present or absent) was almost perfectly concordant with the results of phenotypic susceptibility testing. Various toxin genes were also analyzed successfully. Our analysis demonstrates that using traditional molecular methods and WGS can facilitate the identification of outbreaks and help to control nosocomial transmission.     

中华补血草内生与根际具ACC脱氨酶活性细菌的筛选及其生物多样性

【目的】获得江苏沿海滩涂盐生药用植物中华补血草内生及根际具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的细菌,研究其遗传多样性和潜在促生活性。【方法】从中华补血草和根际土壤分离筛选具有ACC脱氨酶活性的菌株,对其ACC脱氨酶活性定量检测,通过16S r RNA基因序列分析确定菌株系统发育地位。同时研究其固氮、溶磷、产植物生长素吲哚乙酸(IAA)及耐盐能力。【结果】分离筛选获得18株具有ACC脱氨酶活性的内生与根际细菌,定量检测发现其中有13株菌的ACC脱氨酶含量在20 nmolα-KA/(mg Pr·h)以上,有11株菌可以固氮,7株菌能够解磷,9株菌产生IAA。菌株的Na Cl盐耐受范围多数在0–13%之间。16S r RNA基因测序表明,活性菌株分属于7个属,多样性丰富,节杆菌属(Arthrobacter)为优势类群。其中菌株KLBMP 5180为节杆菌属的潜在新种。【结论】江苏沿海滩涂盐生药用植物中华补血草共生环境中具有丰富多样的具ACC脱氨酶活性的菌株,并存在潜在新物种资源,具有进一步研究价值。     

Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH

Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD⁺ concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD⁺ and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I₃₉₅/I₅₅₀), fitting by a single-exponential decay function, can efficiently detect various NAD⁺ levels from 100 to 4000 μM, as well as label NAD⁺/NADH (reduced form of NAD) ratios in the range of 1–50.     

Genomic constitution and taxonomy of the Chinese hexaploids Elymus cylindricus and E. breviaristatus (Poaceae: Triticeae)

Elymus cylindricus (2n = 6x = 42) and E. breviaristatus (2n = 6x = 42) are distributed in grasslands and deserts of northern and north‐western China. Their genomic constitution and taxonomic status are unclear. Elymus cylindricus was crossed with E. wawawaiensis J.R.Carlson & Barkworth ( StH ), Roegneria grandis Keng ( StY ) and Campeiostachys dahurica (Turcz. ex Griseb.) B.R.Baum, J.L. Y ang & C. Y en var. dahurica ( StYH ). Meiotic pairing in the hybrids E. cylindricus × E. wawawaiensis ( StH ), E. cylindricus × R. grandis ( StY ) and E. cylindricus × C. dahurica var. dahurica ( StYH ) showed on average 10.00, 11.30 and 20.92 bivalents per cell, respectively. Elymus breviaristatus was crossed with C. dahurica var. dahurica ( StYH ) and E. cylindricus. Chromosome pairing in the hybrids of E. breviaristatus × C. dahurica var. dahurica and E. breviaristatus × E. cylindricus showed on average 19.60 and 19.27 bivalents, respectively. Genomic in situ hybridization (GI SH ) revealed the presence of St , Y and H genomes in E. cylindricus and E. breviaristatus. An intergenomic rearrangement was observed in E. cylindricus using GI SH . Meiotic pairing data and GI SH indicated that both E. cylindricus and E. breviaristatus are allohexaploids containing the StYH genomes. Elymus cylindricus and E. breviaristatus should be treated as Campeiostachys dahurica var. cylindrica and Campeiostachys breviaristata, respectively.