Background The lateral supramalleolar perforator flap has evolved significantly since its introduction. Initial descriptions focused on its basic vascular anatomy and surgical technique. However, over time, advancements in imaging technology and surgical techniques have led to a deeper understanding of its vascular patterns and potential modifications. Methods To conduct this review, a comprehensive literature search was conducted using electronic databases such as PubMed, Scopus, and Embase. Results The review has highlighted various surgical designs and transfer techniques that have been developed to optimize the flap’s effectiveness in different clinical scenarios. Conclusion This study provides a theoretical basis for further research and the development of lateral supramalleolar perforator flap.

Sterile alpha and Toll/interleukin-1 receptor motif containing 1 (SARM1), a nicotinamide adenine dinucleotide (NAD)-utilizing enzyme, mediates axon degeneration (AxD) in various neurodegenerative diseases. It is activated by nicotinamide mononucleotide (NMN) to produce a calcium messenger, cyclic ADP-ribose (cADPR). This activity is blocked by elevated NAD level. Here, we verified this metabolic regulation in somatic HEK-293T cells by overexpressing NMN-adenyltransferase to elevate cellular NAD, which resulted not only in inhibition of their own SARM1 from producing cADPR but, surprisingly, also in the 5–10 neighboring wildtype cells in mixed cultures via connexin (Cx)-43. Direct visualization of gap junction intercellular communication (GJIC) was achieved by incubating cells with a permeant probe, PC11, which is converted by SARM1 into PAD11, a fluorescent NAD analog capable of traversing GJs. Extending the findings to dorsal root ganglion neurons, we further showed that CZ-48, a permeant NMN analog, or axotomy, activated SARM1 and the produced PAD11 was transferred to contacting axons via GJIC. The gap junction involved was identified as Cx36 instead. This neuronal GJIC was demonstrated to be functional, enabling healthy neurons to protect adjacent axotomized axons from degeneration. Inhibition of GJIC in mice by AAV-PHP.eB-mediated knockdown of Cx36 in brain induced neuroinflammation, which in turn activated SARM1 and resulted in axon degeneration as well as behavioral deficits. Our results demonstrate a novel intercellular regulation mechanism of SARM1 and reveal a protective role of healthy tissue against AxD induced by injury or neuroinflammation.

α-Synuclein is an important drug target for the treatment of Parkinson’s disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5′ untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5′ UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient–derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Neurodegenerative diseases encompass a heterogeneous group of disorders that afflict millions of people worldwide. Characteristic protein aggregates are histopathological hallmark features of these disorders, including Amyloid β (Aβ)-containing plaques and tau-containing neurofibrillary tangles in Alzheimer’s disease, α-Synuclein (α-Syn)-containing Lewy bodies and Lewy neurites in Parkinson’s disease and dementia with Lewy bodies, and mutant huntingtin (mHTT) in nuclear inclusions in Huntington’s disease. These various aggregates are found in specific brain regions that are impacted by neurodegeneration and associated with clinical manifestations. Transglutaminase (TG2) (also known as tissue transglutaminase) is the most ubiquitously expressed member of the transglutaminase family with protein crosslinking activity. To date, Aβ, tau, α-Syn, and mHTT have been determined to be substrates of TG2, leading to their aggregation and implicating the involvement of TG2 in several pathophysiological events in neurodegenerative disorders. In this review, we summarize the biochemistry and physiologic functions of TG2 and describe recent advances in the pathogenetic role of TG2 in these diseases. We also review TG2 inhibitors tested in clinical trials and discuss recent TG2-targeting approaches, which offer new perspectives for the design of future highly potent and selective drugs with improved brain delivery as a disease-modifying treatment for neurodegenerative disorders.

The prognostic significance of radiotherapy (RT) for colorectal cancer (CRC) has shown conflicting results, particularly among different pathological subtypes, including adenocarcinoma (AC), mucinous adenocarcinoma (MC), and signet-ring cell carcinoma (SR). This study analyzed the prognosis of three pathological CRC types and focused on the prognostic significance of RT on three CRC histological subtypes. Patients diagnosed with AC (n = 54,174), MC (n = 3813), and SR (n = 664) in the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) database (2010–2017) were evaluated. Cox regression models and competitive risk models were built to assess the effect of RT on the risk of CRC-associated death. Potential interactions between RT and stratified variables including age, sex, and tumor location were examined by multiplicative models. Compared with AC patients, SR patients had the worst overall survival (OS) among 3 subtypes of CRC (log-rank test, p < 0.001). Compared with patients who did not receive radiotherapy, RT was associated with a 1.09-fold (HR = 1.09, 95%[CI]: 1.03, 1.15) elevated risk of death among AC patients. In the SR group, RT significantly reduced the risk of death by 39% (HR = 0.61, 95%[CI]: 0.39–0.95). However, RT did not appear to independently influence survival in the MC group (HR = 0.96, 95%[CI]: 0.77, 1.21). In the subgroup analysis, tumor location (colon and rectum) significantly modified the association between RT and the risk of death among the AC and SR patients (p for interaction < 0.05). SR patients exhibited a worse OS (overall survival) than AC patients, and the effect of RT varied according to CRC histological subtypes. This can ultimately lead to more personalized and effective treatment strategies for CRC patients.

Prostate cancer is a clinically heterogeneous disease and remains the most common non-skin malignancy in men worldwide (Abate-Shen and Shen, 2000; Litwin and Tan, 2017), which is often diagnosed through screening with digital rectal examinations and quantitation of serum levels of prostate-specific antigen (PSA). At the morphological aspect, the Gleason scoring system is regarded as the most reliable and predictive histological grading system (Welch and Albertsen, 2009; Heidenreich et al., 2011). Interpatient genomic heterogeneity in prostate cancer is well recognized; however, molecular stratification of prostate cancer to guide treatment selection based on predictive genomic biomarkers remains an unmet clinical need (Prensner et al., 2012; Topalian et al., 2016). Developments in next-generation sequencing technologies have increased the speed and reduced the cost of sequencing for cancer samples. And many molecular characterization efforts such as The Cancer Genome Atlas (TCGA) database have unlocked opportunities to characterize the genomic and transcriptomic landscapes of cancer for basic science and clinical oncology research (Ceram‘i et al., 2012). Besides, an enormous amount of omics data from independent prostate cancer studies have been deposited into the NCBI Gene Expression Omnibus (GEO) database (Barrett et al., 2013). The integration of these databases provides an unprecedented opportunity for prostate cancer genomics research. However, an overwhelming amount of multi-omics data from various technical platforms make it increasingly challenging to perform data exploration, analytics, and visualization, especially for scientists without a computational background. Hence, an userfriendly integrated database must be urgently established for the retrieval, integration, and analysis of big data in prostate cancer studies. In this study, we curated the multi-omics data from various data resources and integrated the customized analysis tools to develop an interactive platform, called IPPC, to provide mutations profile, survival probability, gene expression, co-expression, immune infiltration, miRNA–target association, and single-cell sequencing analyses for query genes. We collected a mass of data from a total of 14134 samples in 66 datasets under strict quality control and uniform processing. The IPPC web-server will facilitate the exploration of multidimensional genomics data of prostate cancer by allowing analysis and visualization across genes, samples, and data types. IPPC web-server collected and integrated multi-omics data from public databanks by using the keywords: ‘PCa’, ‘PC’, and ‘prostate cancer’ (before October 30, 2020). To ensure the quality and reliability of data warehousing, we screened, collected, and filtered the data with the following criteria: (i) gene microarray or high-throughput sequencing data extracted from prostate cancer or adjacent normal tissues; (ii) the sample size of each dataset is no less than 50; (iii) for multi-duplicated or same annotated samples, we adapted the quality control to delete the redundant samples; and (iv) the clinical information of samples contains key features: age, race, tumor site, Gleason score, PSA, clinical status, pathologic status, and sample type. In total, we obtained 66 independent datasets including 44 datasets from GEO, 2 datasets from TCGA, and 20 datasets of cBioProtal (Figure 1). We also incorporated the 329 KEGG pathways and 18176 gene ontology terms for gene annotation, and 380639 miRNA–target interactions for gene– miRNA expression association analysis. The dataset screening needs to be affirmed again by another researcher. The quality control was performed on each of the 44 GEO datasets by using the ‘simpleaffy’ and ‘affyPLM’ packages of R software (https://www.r-project.org/ ). The raw data for all the datasets were normalized, summarized, and logtransformed through the robust multiarray average function of ‘affy’ R package. The probe-based expression of genes was converted into gene expression profiles, and the gene containing multiple probes reserved the probes with the largest interquartile ranges while giving up others. For the RNA–seq data, all the samples of the remaining datasets were normalized with fragments per kilobase million (FPKM) or reads per kilobase million (RPKM) and then log-transformed. The somatic mutation data of prostate cancer were filtered to exclude the inappropriate molecular consequences including intergraded variants, non-coding variants, or intron variants and to remain nonsynonymous mutations like missense variants. We compiled a series of clinical features,

Abstract Background: Tim-3 (T-cell immunoglobin and mucin-domain containing-3) has emerged as a promising novel immune checkpoint target playing non-redundant and synergistic roles with PD-1 in triggering T cell exhaustion, however, the complexity of Tim-3-interacting ligands (Gal-9, Ceacam1 and others) has posed a challenge to reconstitute functional Tim-3-targeting immune checkpoint blockers (ICBs) cellular screening system. Materials and methods: To address this challenge, we have engineered Tim-3 Advanced Genomic Manipulated human Jurkat T IL-2-Luc2P reporter cells (Tim-3 AGM T cells) via multiple steps of electroporation-mediated genomic manipulation, and validated the functionality of this reconstituted Tim-3 immune checkpoint cellular system using IL-2-Luc2P reporter assay and IL-2 ELISA following TCR and CD28 engagement. Results: We found that anti-CD3/CD28-induced T cell activation in the Tim-3 AGM T cells as assessed by IL-2-Luc2P reporter was significantly suppressed (~80%) after over-expression of Gal-9 but not the empty vector control, validating functionality of the reconstituted Tim-3/Gal-9 immune checkpoint complex. Conclusions: Such an innovative reconstituted functional Tim-3/Gal-9 immune checkpoint reporter cell line can enable high through-put screening for novel Tim-3 ICBs including small or large molecule therapeutics and following in-depth mechanistic studies. Citation Format: Fang Zhang, Pei Wu, Jun Liu, Baiqiu Wang. Reconstituted functional Tim-3/Gal-9 immune checkpoint cell line to screen for novel Tim-3 ICBs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3258.

SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. We show that CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca2+ messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells.

Hemin is a breakdown product of hemoglobin. It has been reported that the injection of hemin improves lipid metabolism and insulin sensitivity in various genetic models. However, the effect of hemin supplementation in food on lipid metabolism and insulin sensitivity is still unclear, and whether hemin directly affects cellular insulin sensitivity is yet to be elucidated. Here we show that hemin enhances insulin-induced phosphorylation of insulin receptors, Akt, Gsk3β, FoxO1 and cytoplasmic translocation of FoxO1 in cultured primary hepatocytes under insulin-resistant conditions. Furthermore, hemin diminishes the accumulation of triglyceride and increases in free fatty acid content in primary hepatocytes induced by palmitate. Oral administration of hemin decreases body weight, energy intake, blood glucose and triglyceride levels, and improves insulin and glucose tolerance as well as hepatic insulin signaling and hepatic steatosis in male mice fed a high-fat diet. In addition, hemin treatment decreases the mRNA and protein levels of some hepatic genes involved in lipogenic regulation, fatty acid synthesis and storage, and increases the mRNA level and enzyme activity of CPT1 involved in fatty acid oxidation. These data demonstrate that hemin can improve lipid metabolism and insulin sensitivity in both cultured hepatocytes and mice fed a high-fat diet, and show the potential beneficial effects of hemin from food on lipid and glucose metabolism.

Key Laboratory of Nutrition and Metabolism, Chinese Academy of Sciences Center for Excellence in Molecular Cell Sciences, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences (J.L., B.Z., M.Y., R.H., Yu.W., Z.H., Y.Y., C.D., Yi.W., F.Z., Q.Z.), 200031 Shanghai, China; and School of Life Science and Technology (Q.Z.), Shanghai Tech University, Shanghai 200093, China

Circadian misalignment induces insulin resistance in both human and animal models, and skeletal muscle is the largest organ response to insulin. However, how circadian clock regulates muscle insulin sensitivity and the underlying molecular mechanisms are still largely unknown. Here we show circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, two core circadian transcription factors, are down-regulated in insulin-resistant C2C12 myotubes and mouse skeletal muscle. Furthermore, insulin signaling is attenuated in the skeletal muscle of ClockΔ19/Δ19 mice, and knockdown of CLOCK or BMAL1 by small interfering RNAs induces insulin resistance in C2C12 myotubes. Consistently, ectopic expression of CLOCK and BMAL1 improves insulin sensitivity in C2C12 myotubes. Moreover, CLOCK and BMAL1 regulate the expression of sirtuin 1 (SIRT1), an important regulator of insulin sensitivity, in C2C12 myotubes and mouse skeletal muscle, and two E-box elements in Sirt1 promoter are responsible for its CLOCK- and BMAL1-dependent transcription in muscle cells. Further studies show that CLOCK and BMAL1 regulate muscle insulin sensitivity through SIRT1. In addition, we find that BMAL1 and SIRT1 are decreased in the muscle of mice maintained in constant darkness, and resveratrol supplementation activates SIRT1 and improves insulin sensitivity. All these data demonstrate that CLOCK and BMAL1 regulate muscle insulin sensitivity via SIRT1, and activation of SIRT1 might be a potential valuable strategy to attenuate muscle insulin resistance related to circadian misalignment.

It has been reported that some small noncoding RNAs are involved in the regulation of insulin sensitivity. However, whether long noncoding RNAs also participate in the regulation of insulin sensitivity is still largely unknown. We identified and characterized a long noncoding RNA, regulator of insulin sensitivity and autophagy (Risa), which is a poly(A)(+) cytoplasmic RNA. Overexpression of Risa in mouse primary hepatocytes or C2C12 myotubes attenuated insulin-stimulated phosphorylation of insulin receptor, Akt, and Gsk3β, and knockdown of Risa alleviated insulin resistance. Further studies showed that overexpression of Risa in hepatocytes or myotubes decreased autophagy, and knockdown of Risa up-regulated autophagy. Moreover, knockdown of Atg7 or -5 significantly inhibited the effect of knockdown of Risa on insulin resistance, suggesting that knockdown of Risa alleviated insulin resistance via enhancing autophagy. In addition, tail vein injection of adenovirus to knock down Risa enhanced insulin sensitivity and hepatic autophagy in both C57BL/6 and ob/ob mice. Taken together, the data demonstrate that Risa regulates insulin sensitivity by affecting autophagy and suggest that Risa is a potential target for treating insulin-resistance-related diseases.-Wang, Y., Hu, Y., Sun, C., Zhuo, S., He, Z., Wang, H., Yan, M., Liu, J., Luan, Y., Dai, C., Yang, Y., Huang, R., Zhou, B., Zhang, F., Zhai, Q. Down-regulation of Risa improves insulin sensitivity by enhancing autophagy.

CD38, as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein, we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved, identifying three separate epitopes on the carboxyl domain. Chromobodies, engineered by tagging the nanobody with fluorescence proteins, provide fast, simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin, PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines, with half maximal effective concentration reaching as low as 10(-11) molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma.

The protein deacetylase, sirtuin 1 (SIRT1), involved in regulating hepatic insulin sensitivity, shows circadian oscillation and regulates the circadian clock. Recent studies show that circadian misalignment leads to insulin resistance (IR); however, the underlying mechanisms are largely unknown. Here, we show that CLOCK and brain and muscle ARNT‐like protein 1 (BMAL1), two core circadian transcription factors, are correlated with hepatic insulin sensitivity. Knockdown of CLOCK or BMAL1 induces hepatic IR, whereas their ectopic expression attenuates hepatic IR. Moreover, circadian change of insulin sensitivity is impaired in Clock mutant, liver‐specific Bmal1 knockout (KO) or Sirt1 KO mice, and CLOCK and BMAL1 are required for hepatic circadian expression of SIRT1. Further studies show that CLOCK/BMAL1 binds to the SIRT1 promoter to enhance its expression and regulates hepatic insulin sensitivity by SIRT1. In addition, constant darkness‐induced circadian misalignment in mice decreases hepatic BMAL1 and SIRT1 levels and induces IR, which can be dramatically reversed by resveratrol. Conclusion: These findings offer new insights for coordination of the circadian clock and metabolism in hepatocytes by circadian regulation of hepatic insulin sensitivity via CLOCK/BMAL1‐dependent SIRT1 expression and provide a potential application of resveratrol for combating circadian misalignment‐induced metabolic disorders. (Hepatology 2014;59:2196–2206)