Long-term stabilization of stage 4 colon cancer using sodium dichloroacetate therapy

Oral dichloroacetate sodium(DCA) has been investigated as a novel metabolic therapy for various cancers since 2007, based on data from Bonnet et al that DCA can trigger apoptosis of human lung, breast and brain cancer cells. Response to therapy in human studies is measured by standard response evaluation criteria for solid tumours definitions, which define "response" by the degree of tumour reduction, or tumour disappearance on imaging. However, Blackburn et al have demonstrated that DCA can also act as a cytostatic agent in vitro and in vivo, without causing apoptosis(programmed cell death). A case is presented in which oral DCA therapy resulted in tumour stabilization of stage 4 colon cancer in a 57 years old female for a period of nearly 4 years, with no serious toxicity. Since the natural history of stage 4 colon cancer consists of steady progression leading to disability and death, this case highlights a novel use of DCA as a cytostatic agent with a potential to maintain long-term stability of advanced-stage cancer.

Anticancer effect of Psidium guajava(Guava) leaf extracts against colorectal cancer through inhibition of angiogenesis

Objective:To evaluate the anti-angiogenic and anticancer activities of Psidium guajava leaf extracts against angiogenesis-dependent colorectal cancer.Methods:Three extracts were produced using distilled water,ethanol,and n-hexane as solvents.The extracts were physically characterised through gas chromatography–mass spectrometry,ultraviolet–visible spectroscopy,and Fourier transform infrared spectroscopy.Their antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl,total phenolic content,and total flavonoid content assays.To assess their anti-angiogenic activity,cell viability and rat aortic ring assays were conducted,while cell migration,tube formation,colony formation,and VEGF ELISA assays were conducted to elucidate their effects on different aspects of angiogenesis.Molecular docking was used to assess the antiangiogenic potential of some possible compounds in the extracts.Tumour spheroid assay was used to assess the extracts’potential as a treatment for colorectal cancer.Results:The ethanol extract showed the best antioxidant activity.The distilled water and ethanol extracts exhibited more inhibitory activity against EA.hy926 cell viability and aortic ring microvessel growth.In addition,the ethanol extract performed significantly better than the distilled water extract against cell migration and colony formation,and VEGF expression of the cells was suppressed by the ethanol extract.Both the distilled water and ethanol extracts showed significant inhibitory effect on EA.hy926 tube formation and tumour spheroids consisting of EA.hy926 and HCT116 cells.The ethanol extract containedβ-caryophyllene andβ-elemene by phytochemical analysis and subsequent docking studies,which may contribute to its anti-angiogenic activity.Conclusions:The ethanol extract of Psidium guajava has potential in the treatment of colorectal cancer through the inhibition of angiogenesis.

A murine model of tuberculosis/type 2 diabetes comorbidity for investigating the microbiome,metabolome and associated immune parameters

Tuberculosis(TB)is one of the deadliest infectious diseases in the world.The meta-bolic disease type 2 diabetes(T2D)significantly increases the risk of developing ac-tive TB.Effective new TB vaccine candidates and novel therapeutic interventions are required to meet the challenges of global TB eradication.Recent evidence suggests that the microbiota plays a significant role in how the host responds to infection,in-jury and neoplastic changes.Animal models that closely reflect human physiology are crucial in assessing new treatments and to decipher the underlying immunological defects responsible for increased TB susceptibility in comorbid patients.In this study,using a diet-induced murine T2D model that reflects the etiopathogenesis of clinical T2D and increased TB susceptibility,we investigated how the intestinal microbiota may impact the development of T2D,and how the gut microbial composition changes following a very low-dose aerosol infection with Mycobacterium tuberculosis(Mtb).Our data revealed a substantial intestinal microbiota dysbiosis in T2D mice compared to non-diabetic animals.The observed differences were comparable to previous clini-cal reports in TB patients,in which it was shown that Mtb infection causes rapid loss of microbial diversity.Furthermore,diversity index and principle component analyses demonstrated distinct clustering of Mtb-infected non-diabetic mice vs.Mtb-infected T2D mice.Our findings support a broad applicability of T2D mice as a tractable small animal model for studying distinct immune parameters,microbiota and the immune-metabolome of TB/T2D comorbidity.This model may also enable answers to be found to critical outstanding questions about targeted interventions of the gut mi-crobiota and the gut-lung axis.

Advanced pancreatic ductal adenocarcinoma-Complexities of treatment and emerging therapeutic options

Pancreatic ductal adenocarcinoma is a devastating disease with a poor prognosis regardless of stage. To date the mainstay of therapy for advanced disease has been chemotherapy with little incremental im-provements in outcome. Despite extensive research investigating new treatment options the current practices continue to utilise fluorouracil or gemcitabine containing combinations. The need for novel the-rapeutic approaches is mandated by the ongoing poor survival rates associated with this disease. One such approach may include manipulation of ribosome biogenesis and the nucleolar stress response, which has recently been applied to haematological malignancies such as lymphoma and prostate cancer with promising results. This review will focus on the current therapeutic options for pancreatic ductal adenocarcinoma and the complexities associated with developing novel treatments, with a particular emphasis on the role of the nucleolus as a treatment strategy.

Long-term stabilization of metastatic melanoma with sodium dichloroacetate

Sodium dichloroacetate(DCA) has been studied as a metabolic cancer therapy since 2007, based on a publication from Bonnet et al demonstrating that DCA can induce apoptosis(programmed cell death) in human breast, lung and brain cancer cells. Classically, the response of cancer to a medical therapy in human research is measured by Response Evaluation Criterial for Solid Tumours definitions, which define "response" by the degree of tumour reduction, or tumour disappearance on imaging, however disease stabilization is also a beneficial clinical outcome. It has been shown that DCA can function as a cytostatic agent in vitro and in vivo, without causing apoptosis. A case of a 32-year-old male is presented in which DCA therapy, with no concurrent conventional therapy, resulted in regression and stabilization of recurrent metastatic melanoma for over 4 years' duration, with trivial side effects. This case demonstrates that DCA can be used to reduce disease volume and maintain longterm stability in patients with advanced melanoma.

Anti-tumour activity and toxicological studies of combination treatment of Orthosiphon stamineus and gemcitabine on pancreatic xenograft model

BACKGROUND Pancreatic cancer is the most aggressive cancer type.Gemcitabine is the first line chemo-drug used for pancreatic cancer but exerts a broad spectrum of organ toxicities and adverse effects in patients.AIM To evaluate the anti-tumour activity and toxicological effects of Orthosiphon stamineus extract formulation(ID:C5EOSEW5050ESA trademarked as Nuvastatic^(TM)),and gemcitabine combination on pancreatic xenograft model.METHODS Mice were randomly divided into six groups of 6 mice each(n=6)and given different treatments for 28 d.The study design consisted of a 2 x 3 factorial treatment structure,with gemcitabine(yes/no)by oral(at 1200 and 400 mg/kg per day).Human pancreatic cancer cells were injected subcutaneously into the flanks of athymic nude mice.C5EOSEW5050ESA(200 or 400 mg/kg per day)was administered orally,while gemcitabine(10 mg/kg per 3 d)was given intraperitoneally either alone or in combination treatment.Histopathological analyses of vital organs,tumour tissues,and incidence of lethality were analysed.Analyses of tumour necrosis and proliferation were determined by haematoxylin-eosin staining and immunohistochemistry for Ki-67,respectively.RESULTS No signs of toxicity or damage to vital organs were observed in all treatment groups compared to the untreated group.C5EOSEW5050ESA at 200 mg/kg and gemcitabine combination had no additive antitumor effects compared to a single treatment.Remarkably,a comparably greater response in a reduction in tumour growth,Ki-67 protein expression,and necrosis was demonstrated by 400 mg/kg of C5EOSEW5050ESA and gemcitabine combination than that of the individual agents.CONCLUSION These results highlighted the synergistic activity of C5EOSEW5050ESA with gemcitabine to reduce pancreatic tumour growth in mice compared to a single treatment.Thus,this study provides valuable insights into using C5EOSEW5050ESA as a complementary treatment with gemcitabine for pancreatic cancer.

The origin of biological information and programmed protein synthesis

Biological information is one of the most important characteristics of life, and it enables life to evolve to higher complexity and adapt to the environment by mutation and natural selection. However, the origin of this information recording and retrieval system remains a mystery. To understand the origin of biological information will lead us to one step closer to understand the origin of life on earth. Biological information is encoded in DNA and translated into protein by the ribosome in all free living organisms. The information has to be translated into proteins to carry out its biological functions, so the evolution of the ribosome must be integrated with the development of biological information. In this article, I propose that the small ribosomal subunit evolved from a ribozyme that acted as an RNA helicase in the ancient RNA world, and the involvement of tRNAs and the large ribosomal subunit evolved to enhance the helicase activity and to overcome the higher energy require-ment for high GC content RNA helices. This process could have developed as a primitive recording mechanism: since Watson-Crick base paring is a natural property of RNA, each time the proto-small ribosomal subunit came to a particular GC-rich helix, tRNA-like molecules and the proto-large ribosomal subunit would have to be engaged to generate the helicase activity, and consequently the same polypeptide would be synthesized as a by-product. Simple recorded messages then evolved into useful biological information through continuous mutation and natu-ral selection. This hypothesis provides logical and incremental steps for the development of programmed protein synthesis. I also argue that the helicase activity is preserved in the modern ribosome and that from our knowledge of the ribosome, and we can deduce the possible mechanisms of the helicase activity.

利用RAPD-BSA技术筛选小麦耐盐突变位点的分子标记

以耐盐性差的“冀麦 2 4” (TriticumaestivumL .)和其经正定霉素诱变后获得的耐盐突变体 890 1_17为材料 ,用2 80个引物在两者之间进行RAPD分析 ,其中 35个引物扩增出DNA多态性 ,其相似性系数为 0 .978,证明二者为近等基因系 (near_isogenicline ,NIL)。用分株法建立两个F2 群体 (“冀麦 2 4”× 890 1_17和 890 1_17ד中麦 9”) ,在两个群体中按照BSA (bulkedsegregantanalysis)方法分别构建两个对应DNA池 (耐盐池和不耐盐池 ) ,用上述能扩增出明显多态性的 35个引物在对应的耐盐和不耐盐DNA池之间进行RAPD分析 ,发现只有OperonQ4引物在对应的两个DNA池扩增出的多态性在两个F2 群体之间是一致的 ,说明其扩增产物是与耐盐突变位点紧密连锁的RAPD分子标记。

大鼠海马神经干细胞的扩增及与三维微小凹图式复合的研究

目的比较2种培养基下海马神经干细胞(neural stem cells,NSCs)的生长特性,进而实现海马NSCs扩增的优化及其与聚乳酸(poly-L-lactide,PLLA)三维微小凹图式的复合。方法分离大鼠胚胎海马细胞并采用Neurobasal为基础的培养基和DMEM/F12为基础的培养基进行扩增。以四甲基氮唑蓝(MTT)比色法及神经球数目统计法评价2种培养基下细胞增殖行为。采用紫外光光刻、硅蚀刻及软光刻技术制备PLLA三维微小凹图式并实现海马NSCs与微小凹图式的复合。结果原代分离的海马细胞呈神经干细胞标志物阳性并能向神经元系和胶质细胞系分化。在30d的扩增时间内,海马NSCs在以Neurobasal为基础的培养基中缓慢扩增聚集成神经球,少见细胞贴壁及分化;在以DMEM/F12为基础的培养基中海马NSCs扩增迅速,但易贴壁和分化。培养第25天时后者神经球数量为前者的4.7倍。微加工制备的微小凹图式结构清晰、稳定,具有高纵横结构比(≥1)。扩增的海马NSCs能在三维微小凹图式上成功复合生长。结论以DMEM/F12为基础的培养基有利于大鼠海马NSCs的扩增,以Neurobasal为基础的培养基利于海马NSCs的纯化。序贯应用2种培养基可有效扩增海马NSCs并实现其与三维微小凹图式的复合。

Harnessing CRISPR-Cas system diversity for gene editing technologies

The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.

Understand SLE heterogeneity in the era of omics,big data,and artificial intelligence

Systemic lupus erythematosus(SLE)is a systemic autoimmune disease characterized by extraordinary heterogeneity,due to the complex pathogenesis and diverse manifestations.Stratification of patients for therapy and prognosis represents a major challenge to manage SLE.Conventional biomarkers for disease diagnosis and activity assessment provide very limited insight into immunological pathogenesis and therapeutic response rates.The advancement of“omics”technologies including genomics,transcriptomics,proteomics,and metabolomics has constituted an unprecedented opportunity to characterize the immunopathological landscape in individual patients with SLE.Indeed,genomic studies reveal a subset of SLE patients carrying one or more functional single nucleotide polymorphisms(SNPs)underlying immune dysregulation while transcriptomic studies have revealed subgroups in SLE patients showing distinct signatures for Type I interferon(TI-IFN)pathway activation or aberrant differentiation of B cells into plasma cells.This review will summarize results from the latest studies using omics technology to understand SLE heterogeneity.In addition,we propose that the application of artificial intelligence,such as by machine learning-based nonlinear dimensionality reduction method uniform manifold approximation and projection(UMAP)can further strengthen the analysis of omics big data.The combination of new technology and novel analysis pipeline can lead to breakthroughs in stratifying SLE patients for a better monitoring of disease activity and more precise design of treatment regime,not only for conventional immunosuppression but also novel immunotherapies targeting B-cell activating factor(BAFF),TI-IFN,and interleukin 2(IL-2).

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4^(+)T cells

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency(PAD),analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects.CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation.Indeed,while circulating CD57^(+)CD4^(+)T cells are normally rare,we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade.We performed single-cell RNA-seq analysis of matched CD57^(+)CD4^(+)T cells from blood and tonsil samples.Circulating CD57^(+)CD4^(+)T cells(CD4cyt)exhibited a cytotoxic transcriptome similar to that of CD8^(+)effector cells,could kill B cells,and inhibited B-cell responses.CTLA4 restrained the formation of CD4cyt.While CD57 also marked an abundant subset of follicular helper T cells,which is consistent with their antigen-driven differentiation,this subset had a preexhaustion transcriptomic signature marked by TCF7,TOX,and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition.Thus,CD57^(+)CD4^(+)T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers.CTLA4 is a key modifier of CD4^(+)T-cell cytotoxicity,and the pathological CD4cyt phenotype is accentuated by infection.

Delineation of a novel dendritic-like subset in human spleen

Dendritic cells （DCs） and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c^hCD11b＋HLA-DR-hCD86＋ subset in the spleen, along with the previously described subsets of hCDlc＋ DCs, hCD123＋ plasmacytoid DCs （pDCs）, hCD16＋ DCs and hCD141＋ DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c＋HLA-DR＋ and hCD11c＋HLA-DR- cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCDSO and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors.

Multiple checkpoints keep follicular helper T cells under control to prevent autoimmunity

Follicular helper T(Tfh)cells select mutated B cells in germinal centres,which can then differentiate into long-lived high affinity memory B cells and plasma cells.Tfh cells are regulated by a unique molecular programme orchestrated by the transcriptional repressor Bcl6.This transcription factor turns down expression of multiple genes,including transcriptional regulators of other T helper lineages and a vast amount of microRNAs.This enables Tfh cells to express a suite of chemokine receptors,stimulatory ligands and cytokines that enable migration into B-cell follicles,and provision of effective help to B cells.Not surprisingly,dysregulation of this powerful helper subset can lead to a range of autoantibody-mediated diseases;indeed,aberrant accumulation of Tfh cells has been linked with systemic lupus erythematosus,Sjogren’s disease and autoimmune arthritis.Here we dissect multiple checkpoints that operate throughout Tfh cell development and maturation to maintain immunological tolerance while mounting robust and long-lasting antibody responses.

DOCK8 regulates signal transduction events to control immunity

Genetic mutations in the gene encoding DOCK8 cause an autosomal recessive form of hyper immunoglobulin E syndrome （AR-HIES）, referred to as DOCK8 deficiency. DOCK8 deficiency in humans results in the onset of combined immunodeficiency disease （CID）, clinically associated with chronic infections with diverse microbial pathogens, and a predisposition to malignancy. It is now becoming clear that DOCK8 regulates the function of diverse immune cell sub-types, particularly lymphocytes, to drive both innate and adaptive immune responses. Early studies demonstrated that DOCK8 is required for lymphocyte survival, migration and immune synapse formation, which translates to poor pathogen control in the absence of DOCK8. However, more recent advances have pointed to a crucial role for DOCK8 in regulating the signal transduction events that control transcriptional activity, cytokine production and functional polarization of immune cells. Here, we summarize recent advances in our understanding of DOCK8 function, paying particular attention to an emerging role as a signaling intermediate to promote immune responses to diverse external stimuli.

An optimized method to differentiate mouse follicular helper T cells in vitro

Upon priming,naive CD4^(+) helper T(Th)cells differentiate into distinct subsets with specialized functions.The differentiation of Th subsets is driven not only by signals from the T cell receptor(TCR)and costimulatory receptors but is also critically dependent on the specific cytokine milieu.By mimicking such conditions,robust methods have been developed for the in vitro differentiation of type 1 and type 2 Th(Th1 and Th2)cells,and more recently,IL-17-producing Th(Th17)cells and regulatory T(Treg)cells,1 which greatly support the research and applications of these Th subsets.Follicular helper T(Tfh)cells represent another Th subset that specializes in supporting the germinal center(GC)response and regulating the generation of memory B cells and long-lived plasma cells.2 However,current methods for in vitro Tfh differentiation are not optimal.Even in the best practice,only 20%of polarized cells showed the expression of CXCR5,the key Tfh functional marker.3 Here,we report an optimized in vitro differentiation method that generates 50–75%CXCR5+cells with enhanced B cell helper function.We demonstrate that the priming of antigen-presenting cells(APCs)by lipopolysaccharide(LPS)and the increase of the APC:T cell ratio were key to efficiently generating Tfh cells in vitro.

Immunophenotyping identifies distinct cellular signatures for systemic lupus erythematosus and lupus nephritis

Background:Systemic lupus erythematosus(SLE)is a complex systemic autoimmune disease characterized by development of autoantibodies and multiorgan involvement.Kidney involvement,termed lupus nephritis,has major impact on life expectancy.It is increasingly recognized that SLE is likely a common clinical manifestation of pathophysiologically diverse processes,and lupus nephritis has similarly been associated with several distinct immunological processes.We compared the immune cell phenotypes of individuals with SLE in the presence or absence of nephritis.Methods:Cryopreserved peripheral blood mononuclear cells from SLE patients with and without kidney involvement underwent flow cytometric analysis to identify major populations in T cells,B cells and myeloid lineages.Results:We compared the frequencies of lymphocyte populations in 69 SLE patients without nephritis,20 SLE patients with nephritis,and 92 healthy blood donors.Patients with SLE and lupus nephritis(LN)had reduced marginal zone B cells(P<0.0001 in SLE;P=0.001 in LN),memory B cells(P=0.002 in SLE;P=0.001 in LN)and circulating T follicular helper(Tfh)memory cells(P<0.0001 in SLE and LN)compared to healthy donors.Patients with lupus nephritis had increase Th2(P<0.0001)and T regulatory cells(P<0.0001)compared to both SLE patients without nephritis and healthy donors.Conclusion:SLE patients with and without lupus nephritis have distinct immunologic differences that may reflect the unique pathophysiological processes contributing to disease manifestations.