Institute of Life Sciences

Given the marginal penetration of most drugs across the blood-brain barrier, the efficacy of various agents remains limited for glioblastoma (GBM). Here we employ low-intensity pulsed ultrasound (LIPU) and intravenously administered microbubbles (MB) to open the blood-brain barrier and increase the concentration of liposomal doxorubicin and PD-1 blocking antibodies (aPD-1). We report results on a cohort of 4 GBM patients and preclinical models treated with this approach. LIPU/MB increases the concentration of doxorubicin by 2-fold and 3.9-fold in the human and murine brains two days after sonication, respectively. Similarly, LIPU/MB-mediated blood-brain barrier disruption leads to a 6-fold and a 2-fold increase in aPD-1 concentrations in murine brains and peritumoral brain regions from GBM patients treated with pembrolizumab, respectively. Doxorubicin and aPD-1 delivered with LIPU/MB upregulate major histocompatibility complex (MHC) class I and II in tumor cells. Increased brain concentrations of doxorubicin achieved by LIPU/MB elicit IFN-γ and MHC class I expression in microglia and macrophages. Doxorubicin and aPD-1 delivered with LIPU/MB results in the long-term survival of most glioma-bearing mice, which rely on myeloid cells and lymphocytes for their efficacy. Overall, this translational study supports the utility of LIPU/MB to potentiate the antitumoral activities of doxorubicin and aPD-1 for GBM.

Background In recent decades, phytotherapy has remained as a key therapeutic option for the treatment of various cancers. Evodiamine, an excellent phytocompound from Evodia fructus, exerts anticancer activity in several cancers by modulating drug resistance. However, the role of evodiamine in cisplatin-resistant NSCLC cells is not clear till now. Therefore, we have used evodiamine as a chemosensitizer to overcome cisplatin resistance in NSCLC. Methods Here, we looked into SOX9 expression and how it affects the cisplatin sensitivity of cisplatin-resistant NSCLC cells. MTT and clonogenic assays were performed to check the cell proliferation. AO/EtBr and DAPI staining, ROS measurement assay, transfection, Western blot analysis, RT-PCR, Scratch & invasion, and comet assay were done to check the role of evodiamine in cisplatin-resistant NSCLC cells. Results SOX9 levels were observed to be higher in cisplatin-resistant A549 (A549CR) and NCI-H522 (NCI-H522CR) compared to parental A549 and NCI-H522. It was found that SOX9 promotes cisplatin resistance by regulating β-catenin. Depletion of SOX9 restores cisplatin sensitivity by decreasing cell proliferation and cell migration and inducing apoptosis in A549CR and NCI-H522CR. After evodiamine treatment, it was revealed that evodiamine increases cisplatin-induced cytotoxicity in A549CR and NCI-H522CR cells through increasing intracellular ROS generation. The combination of both drugs also significantly inhibited cell migration by inhibiting epithelial to mesenchymal transition (EMT). Mechanistic investigation revealed that evodiamine resensitizes cisplatin-resistant cells toward cisplatin by decreasing the expression of SOX9 and β-catenin. Conclusion The combination of evodiamine and cisplatin may be a novel strategy for combating cisplatin resistance in NSCLC. Graphical abstract

Effective treatments for stroke after the acute phase remain elusive. Muse cells are endogenous, pluripotent, immune-privileged stem cells capable of selectively homing to damaged tissue after intravenous injection and replacing damaged/lost cells via differentiation. This randomized, double-blind, placebo-controlled trial enrolled ischemic stroke patients with modified Rankin Scale (mRS) ≥3. Randomized patients received a single intravenous injection of an allogenic Muse cell-based product, CL2020 (n = 25), or placebo (n = 10), without immunosuppressant, 14–28 days after stroke onset. Safety (primary endpoint: week 12) and efficacy (mRS, other stroke-specific measures) were assessed up to 52 weeks. Key efficacy endpoint was response rate (percentage of patients with mRS ≤2 at week 12). To week 12, 96% of patients in the CL2020 group experienced adverse events and 28% experienced adverse reactions (including one Grade 4 status epilepticus), compared with 100% and 10%, respectively, in the placebo group. Response rate was 40.0% (95% CI, 21.1–61.3) in the CL2020 group and 10.0% (0.3–44.5) in the placebo group; the lower CI in the CL2020 group exceeded the preset efficacy threshold (8.7% from registry data). This randomized placebo-controlled trial demonstrated CL2020 is a possible effective treatment for subacute ischemic stroke. Registry information: JAPIC Clinical Trials Information site (JapicCTI-184103, URL: https://www.clinicaltrials.jp/cti-user/trial/ShowDirect.jsp?japicId=JapicCTI-184103 ).

Periodontal disease and arthroplasty prosthesis loosening and destabilization are both associated with osteolysis, which is predominantly caused by abnormal bone resorption triggered by pro-inflammatory cytokines. Osteoclasts (OCs) are critical players in the process. Concerns regarding the long-term efficacy and side effects of current frontline therapies, however, remain. Alternative therapies are still required. The aim of this work was to investigate the involvement of Tenacissoside H (TDH) in RANKL-mediated OC differentiation, as well as inflammatory osteolysis and associated processes. In vitro, bone marrow-derived macrophages (BMMs) cultured with RANKL and M-CSF were used to detect TDH in the differentiation and function of OCs. Real-time quantitative PCR was used to measure the expression of specific genes and inflammatory factors in OCs. Western blot was used to identify NFATc1, IKK, NF-κB, MAPK pathway, and oxidative stress-related components. Finally, an LPS-mediated calvarial osteolysis mouse model was employed to explore TDH's role in inflammatory osteolysis. The results showed that in vivo TDH inhibited the differentiation and resorption functions of OCs and down-regulated the transcription of osteoclast-specific genes, as well as Il-1β, Il-6 and Tnf-α. In addition, TDH inhibited the IKK and NF-κB signalling pathways and down-regulated the level of ROS. In vivo studies revealed that TDH improves the bone loss caused by LPS. TDH may be a new candidate or treatment for osteoclast-associated inflammatory osteolytic disease.

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell–cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

Objectives: Human thyroglobulin (Tg) is widely used as a tumor marker for recurrence and metastasis of differentiated thyroid cancer (DTC). Currently, serum Tg values are measured using second-generation sandwich immunoassays (2nd-IMA). However, interference by endogenous autoantibodies to thyroglobulin (TgAbs) can lead to false-negative results or falsely low Tg values. Here, we describe a new Tg assay using the immunoassay for total antigen including complex via pretreatment (iTACT) method to prevent TgAb interference and compare it with 2nd-IMA. Methods: Tg values were evaluated by three assays: iTACT Tg, Elecsys Tg-II, which is a 2nd-IMA, and LC-MS/MS (Liquid chromatography tandem-mass spectrometry). The ratio of Tg values between each assay was then compared to the Tg value by LC-MS/MS and TgAb titer. Tg immunoreactivity was analyzed by size-exclusion chromatography. Results: Correlation between iTACT Tg and LC-MS/MS using TgAb-positive specimens was good: Passing-Bablok regression with iTACT Tg = 1.084 x LC-MS/MS + 0.831. Correlation between 2nd-IMA and LC-MS/MS showed a relatively lower slope: 2nd-IMA = 0.747 x LC-MS/MS - 0.518. Thus, Tg values determined by iTACT Tg are equivalent to those of LC-MS/MS regardless of TgAb titer, whereas 2nd-IMA gave lower Tg values due to TgAb interference. Tg-TgAb complexes of various molecular weights were verified by size-exclusion chromatography. Tg values measured by 2nd-IMA fluctuated depending on the molecular weight of the Tg-TgAb complexes, whereas iTACT Tg accurately quantified Tg values regardless of the size of the Tg-TgAb complexes. Conclusion: Tg values in TgAb-positive specimens were accurately determined by iTACT Tg. TgAb-positive specimens contain Tg-TgAb complexes of various molecular weights that interfere with Tg value determination by 2nd-IMA, whereas iTACT Tg is unaffected by the presence of Tg-TgAb complexes.

This study highlights the development of a lab-scale, indigenously designed; Packed-Bed Biofilm Reactor (PBBR) packed with brick pieces. The developed biofilm in the reactor was used for the decolourisation and biodegradation of the textile industry effluent. The PBBR was continuously operated for 264 days, during which 301 cycles of batch and continuous treatment were operated. In batch mode under optimised conditions, more than 99% dye decolourisation and ≥ 92% COD reduction were achieved in 6 h of contact time upon supplementation of effluent with 0.25 g L−1 glucose, 0.25 g L−1 urea, and 0.1 g L−1 phosphates. A decolourisation rate of 133.94 ADMI units h−1 was achieved in the process. PBBR, when operated in continuous mode, showed ≥ 95% and ≥ 92% reduction in ADMI and COD values. Subsequent aeration and passage through the charcoal reactor assisted in achieving a ≥ 96% reduction in COD and ADMI values. An overall increase of 81% in dye-laden effluent decolourisation rate, from 62 to 262 mg L−1 h−1, was observed upon increasing the flow rate from 18 to 210 mL h−1. Dye biodegradation was determined by UV–Vis and FTIR spectroscopy and toxicity study. SEM analysis showed the morphology of the attached-growth biofilm.

Mitochondria are major sources of cytotoxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, that when uncontrolled contribute to cancer progression. Maintaining a finely tuned, healthy mitochondrial population is essential for cellular homeostasis and survival. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating damaged ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. E3 ubiquitin ligase Gp78 is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume, elevated ROS production and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Xenograft tumors of HT-1080 knockout cells show increased labeling for mitochondria and the cell proliferation marker Ki67 and reduced labeling for the TUNEL cell death reporter. Basal Gp78-dependent mitophagic flux is, therefore, selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population, limiting ROS production and tumor cell proliferation.

Stem cells undergo cytokine-driven differentiation, but this process often takes longer than several weeks to complete. A novel mechanism for somatic stem cell differentiation via phagocytosing ‘model cells’ (apoptotic differentiated cells) was found to require only a short time frame. Pluripotent-like Muse cells, multipotent mesenchymal stem cells (MSCs), and neural stem cells (NSCs) phagocytosed apoptotic differentiated cells via different phagocytic receptor subsets than macrophages. The phagocytosed-differentiated cell-derived contents (e.g., transcription factors) were quickly released into the cytoplasm, translocated into the nucleus, and bound to promoter regions of the stem cell genomes. Within 24 ~ 36 h, the cells expressed lineage-specific markers corresponding to the phagocytosed-differentiated cells, both in vitro and in vivo. At 1 week, the gene expression profiles were similar to those of the authentic differentiated cells and expressed functional markers. Differentiation was limited to the inherent potential of each cell line: triploblastic-, adipogenic-/chondrogenic-, and neural-lineages in Muse cells, MSCs, and NSCs, respectively. Disruption of phagocytosis, either by phagocytic receptor inhibition via small interfering RNA or annexin V treatment, impeded differentiation in vitro and in vivo. Together, our findings uncovered a simple mechanism by which differentiation-directing factors are directly transferred to somatic stem cells by phagocytosing apoptotic differentiated cells to trigger their rapid differentiation into the target cell lineage.

Cancer therapy has achieved new heights with numerous advancements made in this field still, cancer treatment modalities including chemotherapy, radiotherapy, immunotherapy, neo-adjuvant therapy, and surgery have limitations in clinics and urges for new therapeutics. Natural products remain the mainstay of chemotherapy since ancient times. Flavonoids are a class of readily available polyphenolic secondary plant metabolites with remarkable anti-cancerous properties. However, poor aqueous solubility, rapid clearance from the blood, and poor bioavailability hinder their clinical use. In this direction, advancements in nanoparticle synthesis and functionalization have produced nanoscale drug delivery agents affording safe and target-specific delivery. Here, we highlight the importance of nanotechnology in safely delivering flavonoids to tumor tissues with high efficiency. We have also described the advancement of flavonoid-loaded nanoparticles either single or in combination with a conventional chemotherapeutic in pre-clinical settings which distinguishes them from other chemotherapeutics. While enormous pre-clinical studies have been published on flavonoid-based nanoparticles the emphasis here is to push these nanomedicines into clinical studies which are likely to influence clinical investigations and their translation for the treatment of cancer patients.

Drug delivery to central nervous system (CNS) diseases is one of the most challenging tasks. The innate blood-brain barrier (BBB) and the blood-cerebrospinal fluid (BCSF) barrier create an obstacle for effective systemic drug delivery to the CNS, by limiting the access of drugs to the brain. Nanotechnology-based drug delivery platform offers a potential therapeutic approach for the treatment of neurological disorders. Several studies have shown that nanomaterials have great potential to be used for the treatment of CNS diseases. The nanocarriers have simplified targeted delivery of therapeutics into the brain by surpassing the BBB and actively inhibiting the disease progression of CNS disorders. The review is an overview of the recent developments in nanotechnology-based drug delivery approaches for major CNS diseases like Alzheimer's disease, Parkinson's disease, ischemic stroke, and Glioblastoma. This review discusses the disease biology of major CNS disorders describing various nanotechnology-based approaches to overcome the challenges associated with CNS drug delivery, focussing on nanocarriers in preclinical and clinical studies for the same. The review also sheds light on the challenges during clinical translation of nanomedicine from bench to bedside. Conventional therapeutic agents used for the treatment of CNS disorders are inadequate due to their inability to cross BBB or BCSF, higher efflux from BBB, related toxicity, and poor pharmacokinetics. The amalgamation of nanotechnology with conventional therapeutic agents can greatly ameliorate the pharmacokinetic associated problems and at the same time assist efficient delivery to CNS.

Background Although still considered a safer alternative to classical cigarettes, growing body of work points to harmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect of e-cigarettes needs to be investigated in more detail considering their widespread use. Methods In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, with and without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cellular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performed via high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry. Results E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however it negatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletion in total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteins were upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of posttranslational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data are available via ProteomeXchange with identifier PXD032071. Conclusions Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMs alterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.

Silent mating type information regulation 2 homolog 1 (SIRT1) is an NAD ⁺ dependent class III histone deacetylase (HDAC) which plays a vital role in cell survival, senescence and metabolism. SIRT1 is overexpressed in several cancers, including breast cancer. Due to the lack of endocrine therapy for ER-ve breast cancers, it is essential to identify the molecular targets having therapeutic importance. SIRT1 is a known target gene of estrogen receptor α (ERα) and well associated with ERα deacetylation. Transcription factor Estrogen related receptors (ERRs) share sequence homology with ERs in DNA binding domain, therefore possibility of sharing target genes between them is high. Our present study aim to elucidate the role of ERRβ in the regulation of SIRT1 in breast cancer tumorigenesis. SIRT1 was highly up-regulated in ER + ve breast cancer cells and tissues. Analysis of 5’ upstream region of SIRT1 suggested the presence of six putative ERRE sites and in-vitro and in-vivo studies confirmed the binding of ERRβ to them. We found SIRT1 to be up-regulated in ERRβ overexpressed ER + ve breast cancer cells. Furthermore, our results suggested the up-regulation of SIRT1 upon ectopic expression of ERRβ along with PCAF. We also proved the tumorigenic role of SIRT1 with enough evidence showing the significant role of SIRT1 in cell proliferation, migration and colony formation capacity. Collectively our results suggest the tumorigenic role of SIRT1 in the breast cancer and SIRT1 inhibitors might serve as potential therapeutic drugs for the treatment of breast cancer.

Circulating tumor DNA (ctDNA) correlates with tumor burden and provides early detection of treatment response and tumor genetic alterations in breast cancer. Neoadjuvant chemotherapy (NACT) has become standard therapy for local advanced breast cancer (LABC). The aim of our study was to investigate plasma ctDNA as a prognostic marker for outcome in patients with LABC treated with NACT. A total of 56 patients with LABC were involved in this study. ctDNA mutations were investigated by using a 100 gene panel-target capture next-generation sequencing. The patients then received standard NACT therapy: adriamycin and cyclophosphamide and paclitaxel (AC-T) or AC-TH (AC-T+ Trastuzumab) regimen. The efficacy of NACT was evaluated by Miller-Payne grading system. A predictive and weight model was used to screen ctDNA point mutation biomarkers for NACT. The ctDNA mutational profile of LABC patients was identified. For nonsynonymous mutations, the top 5 mutated genes were MTHFR (51/56, 91.1%), XPC (50/56, 89.3%), ABCB1 (48/51, 94.1%), BRCA2 (38/56, 67.9%), and XRCC1 (38/56, 67.9%). In addition, the mutation frequencies of PIK3CA and TP53 were 32.1% (18/56) and 26.8% (15/56), respectively. The predictive model indicated that XRCC1 44055726 (TG>-) mutation (25/56, 44.6%) was significantly associated with Miller-Payne 4-5 and Miller-Payne 3-5 responses. While mTOR 11249132(G>C) mutation (23/56, 41.1%) was associated with Miller-Payne 1-4 or Miller-Payne 1-3 responses. Furthermore, XRCC1 44055726 (TG>-) accompanied by mTOR wild type predicted a good NACT efficacy in all response classification systems. The ROC curves to discriminate good neoadjuvant chemotherapy efficiency (Miller-Payne 4-5) and poor efficiency (Miller-Payne 1-3) were created, and AUC value was 0.77. Our results suggested that ctDNA mutation of XRCC1 44055726 (TG>-) might be a positive biomarker for NACT therapy in LABC, while mTOR 11249132(G>C) mutation was potentially associated with NACT resistance.

The demand for rice is likely to increase approximately 1.5 times by the year 2050. In contrast, the rice production is stagnant since the past decade as the ongoing rice breeding program is unable to increase the production further, primarily because of the problem in grain filling. Investigations have revealed several reasons for poor filling of the grains in the inferior spikelets of the compact panicle, which are otherwise genetically competent to develop into well-filled grains. Among these, the important reasons are 1) poor activities of the starch biosynthesizing enzymes, 2) high ethylene production leading to inhibition in expressions of the starch biosynthesizing enzymes, 3) insufficient division of the endosperm cells and endoreduplication of their nuclei, 4) low accumulation of cytokinins and indole-3-acetic acid (IAA) that promote grain filling, and 5) altered expressions of the miRNAs unfavorable for grain filling. At the genetic level, several genes/QTLs linked to the yield traits have been identified, but the information so far has not been put into perspective toward increasing the rice production. Keeping in view the genetic competency of the inferior spikelets to develop into well-filled grains and based on the findings from the recent research studies, improving grain filling in these spikelets seems plausible through the following biotechnological interventions: 1) spikelet-specific knockdown of the genes involved in ethylene synthesis and overexpression of β-CAS (β-cyanoalanine) for enhanced scavenging of CN⁻ formed as a byproduct of ethylene biosynthesis; 2) designing molecular means for increased accumulation of cytokinins, abscisic acid (ABA), and IAA in the caryopses; 3) manipulation of expression of the transcription factors like MYC and OsbZIP58 to drive the expression of the starch biosynthesizing enzymes; 4) spikelet-specific overexpression of the cyclins like CycB;1 and CycH;1 for promoting endosperm cell division; and 5) the targeted increase in accumulation of ABA in the straw during the grain filling stage for increased carbon resource remobilization to the grains. Identification of genes determining panicle compactness could also lead to an increase in rice yield through conversion of a compact-panicle into a lax/open one. These efforts have the ability to increase rice production by as much as 30%, which could be more than the set production target by the year 2050.

Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1 cre knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1 cre -containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout β-cells from female, but not male mice, whereas only male β Insr KO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female β Insr KO and β Insr HET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p . glucose challenge. Deletion of Insr does not alter β-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include β-cell insulin resistance, which predicts that β-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female β Insr KO and β Insr HET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1 -CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that β-cell insulin resistance in the form of reduced β-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

Aims: To prevent hepatitis B virus (HBV) reactivation-related hepatitis, we examined the clinical usefulness of a highly-sensitive HB core-related antigen (iTACT-HBcrAg) assay in patients with resolved HBV infection after nucleos(t)ide analogues (NA) treatment for HBV reactivation. Methods: We retrospectively analyzed 27 patients with resolved HBV infection who experienced HBV reactivation (defined as HBV DNA levels of 1.3 log IU/mL or more), and who received systemic chemotherapies for hematological malignancies between 2008 and 2020. iTACT-HBcrAg, HBsAg-HQ and anti-HBs were measured using samples stored after HBV reactivation. The lower limit of quantification for iTACT-HBcrAg was 2.0 log U/mL. Results: HBV reactivation was diagnosed at a median HBV DNA level of 1.8 log IU/mL, and then all patients received NA treatment. No patient had HBV-related hepatitis with a median maximum HBV DNA level of 2.0 log IU/mL. The positivities of iTACT-HBcrAg and HBsAg-HQ was 96% and 52% after HBV reactivation, respectively. Of 25 patients with detectable iTACT-HBcrAg at the initiation of NA treatment, 17 (68%) achieved iTACT-HBcrAg loss. Median durations from NA treatment to HBV DNA loss and iTACT-HBcrAg loss or the last follow-up were 35 and 175 days, respectively. Recurrence of HBV reactivation after NA cessation was not observed in seven of eight patients who achieved iTACT-HBcrAg loss or seropositive for anti-HBs during follow-up, except for one without anti-HBs after allogeneic transplantation. Conclusions: iTACT-HBcrAg could be a potential surrogate marker for diagnosing early-stage HBV reactivation as well as safe cessation of NA treatment in patients with resolved HBV infection after HBV reactivation. This article is protected by copyright. All rights reserved.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.