Targeting Solute Carrier Transporters (SLCs) as a Therapeutic Target in Different Cancers.

Solute carrier (SLC) transporters constitute a vast superfamily of transmembrane proteins tasked with regulating the transport of various substances such as metabolites, nutrients, ions, and drugs across cellular membranes. SLC transporters exhibit coordinated expression patterns across normal tissues, suggesting a tightly regulated regulatory network governing normal cellular functions. These transporters are crucial for the transport of various metabolites, including carbohydrates, proteins, lipids, and nucleic acids. However, during tumor development, metabolic changes drive an increased demand for energy and nutrients. Consequently, tumor cells alter the expression of SLC transporters to meet their heightened nutrient requirements. Targeting SLCs through inhibition or activation presents a promising therapeutic approach in cancer treatment. Certain SLCs also serve as intriguing chemo-sensitizing targets, as modulating their activity can potentially alter the response to chemotherapy. This review underscores the significance of various SLCs in tumor progression and underscores their potential as both direct and indirect targets for cancer therapy.

Synthesis and validation of click-modified NOD1/2 agonists.

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

What are NLRP3-ASC specks? an experimental progress of 22 years of inflammasome research.

Speck assembly is the hallmark of NLRP3 inflammasome activation. The 1µm structure comprising of NLRP3 and ASC is the first observable phenotype of NLRP3 activation. While the common consensus is that the specks are the site of inflammasome activity, no direct experimental evidence exists to support this notion. In these 22 years, since the inflammasome discovery, several research studies have been published which directly or indirectly support or refute the idea of speck being the inflammasome. This review compiles the data from two decades of research to answer a long-standing question: "What are NLRP3-ASC specks?"

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

Dextran/eudragit S-100 based redox sensitive nanoparticles for colorectal cancer therapy.

Herein, we present disulfide crosslinked dextran/eudragit S-100 nanoparticles (DEEU NPs) (≈55 nm) for colorectal cancer treatment. These redox environment sensitive DEEU NPs are synthesized by simple oxidation of thiolated polymers in air. This approach allows avoiding the use of any additional chemical crosslinker. These DEEU NPs have high encapsulation efficiency for the doxorubicin (DOX) model drug (≈95%). The prepared DEEU NPs are redox sensitive owing to disulfide units and exhibit ≈80% DOX release in the reducing environment of GSH. Additionally, DOX-DEEU NPs display enhanced cytotoxicity for HCT116 cancer cells as compared to free DOX. Annexin V staining results also support the higher anticancer efficiency of DOX-DEEU NPs via induction of apoptosis. In vivo biodistribution results demonstrate that DEEU NPs can remain in the colon region for up to 24 hours. These results indicate that DEEU NPs can act as a promising platform for colorectal cancer treatment.

Intraoperative portal vein stenting through umbilical vein approach: An innovative salvage procedure for portal vein thrombosis in pediatric liver transplant.

BACKGROUND: IPVS is considered a last resort or a salvage procedure in the event of recurrent PV thrombosis despite multiple attempts at redo PV anastomosis. We employed the opened umbilical vein approach to place the stent in the PV and deliver anticoagulation through a catheter. MATERIALS AND METHODS: From Jan 2017 to Feb 2022, 150 patients underwent pediatric transplantation at department of liver transplant and hepatobiliary surgery unit, Indraprastha Apollo hospitals, New Delhi. Age, weight, PELD Score, diagnosis, portal vein diameter on preoperative CT, Portal flow after stenting, decrease in spleen size after stenting in follow-up CT were collected from a prospectively maintained data base and reviewed. RESULTS: Eight patients underwent IPVS following LDLT (mean age-10.6 ± 2.2 months, mean weight 8.1 ± 1.6, mean PELD score 32.7 ± 7.3). The mean PV diameter on preoperative CT scan was 3.6 mm (range 2.7-5.6 mm). The mean portal flow following stenting was 718.75 cc/min. Percentage reduction in size of the spleen was 26.35% beyond 2nd post-operative week. No patient had recurrent PV thrombosis following IPVS and all maintained an adequate portal flow throughout the immediate postoperative period. Two patients had in-hospital mortality secondary to septic complications. CONCLUSION: Umbilical vein approach is technically feasible, easy to manipulate the stent and catheter placement after stenting helps to deliver anticoagulants locally.

Biodegradable disulfide crosslinked chitosan/stearic acid nanoparticles for dual drug delivery for colorectal cancer.

Herein, redox responsive chitosan/stearic acid nanoparticles (CSSA NPs) (≈200 nm) are developed for dual drug delivery. These degradable nanoparticles are prepared based on disulfide (SS) crosslinking chemistry avoiding the use of any external crosslinking agent. CSSA NPs are further loaded with both DOX (hydrophilic) and curcumin (hydrophobic) drugs with ≈86 % and ≈82 % encapsulation efficiency respectively. This approach of combining anticancer therapeutics having different mode of anticancer action allows to develop systems for cancer therapy with enhanced efficacy. In vitro drug release experiments clearly exhibit the low leakage of drug under physiological conditions while ≈98 % DOX and ≈96 % curcumin is released after 136 h under GSH reducing conditions. The cytotoxicity experiments against HCT116 cells demonstrate higher cytotoxicity of dual drug loaded CSSA NPs. In vivo biodistribution experiments with c57bl/6j mice confirms the retention of CSSA NPs in the colon area up to 24 h exhibiting their potential for colorectal cancer therapy.

Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder.

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is a bifunctional enzyme (N-terminal epimerase and C-terminal Kinase domain) that catalyses the rate limiting step in sialic acid biosynthesis. More than 200 homozygous missense or compound heterozygous mutations in GNE have been reported worldwide to cause a rare neuromuscular disorder, GNE myopathy. It is characterized by a slowly progressive defect in proximal and distal skeletal muscles with patients becoming wheel-chair-bound. There are no current approved therapies available for GNE myopathy. ManNAc therapy is currently in advanced clinical trials and has shown signs of slowing the disease progression in a phase 2 trial. The present study aims to understand the effect of GNE mutation on its enzymatic activity and identification of potential small effector molecules. We characterized different GNE mutations (p.Asp207Val, p.Val603Leu, p.Val727Met, p.Ile618Thr and p.Arg193Cys) prevalent in Asian population that were cloned, expressed and purified from Escherichia coli as full-length recombinant proteins. Our study demonstrates that full length GNE can be expressed in E. coli in its active form and analysed for the functional activity. Each mutation showed variation in epimerase and kinase activity and responded to the small effector molecules (metformin, BGP-15 kaempferol, catechin, quercetin) in a differential manner. Our study opens an area for futuristic structural determination of full length GNE and identification of potential therapeutic molecules.

Macrophages as a Potential Immunotherapeutic Target in Solid Cancers.

The revolution in cancer immunotherapy over the last few decades has resulted in a paradigm shift in the clinical care of cancer. Most of the cancer immunotherapeutic regimens approved so far have relied on modulating the adaptive immune system. In recent years, strategies and approaches targeting the components of innate immunity have become widely recognized for their efficacy in targeting solid cancers. Macrophages are effector cells of the innate immune system, which can play a crucial role in the generation of anti-tumor immunity through their ability to phagocytose cancer cells and present tumor antigens to the cells of adaptive immunity. However, the macrophages that are recruited to the tumor microenvironment predominantly play pro-tumorigenic roles. Several strategies targeting pro-tumorigenic functions and harnessing the anti-tumorigenic properties of macrophages have shown promising results in preclinical studies, and a few of them have also advanced to clinical trials. In this review, we present a comprehensive overview of the pathobiology of TAMs and their role in the progression of solid malignancies. We discuss various mechanisms through which TAMs promote tumor progression, such as inflammation, genomic instability, tumor growth, cancer stem cell formation, angiogenesis, EMT and metastasis, tissue remodeling, and immunosuppression, etc. In addition, we also discuss potential therapeutic strategies for targeting TAMs and explore how macrophages can be used as a tool for next-generation immunotherapy for the treatment of solid malignancies.

An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1.

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica and have described some of the molecules that play key roles in the process. Here we showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica.

Coordinated activity of amoebic formin and profilin are essential for phagocytosis.

For the protist parasite Entamoeba histolytica, endocytic processes, such as phagocytosis, are essential for its survival in the human gut. The actin cytoskeleton is involved in the formation of pseudopods and phagosomal vesicles by incorporating a number of actin-binding and modulating proteins along with actin in a temporal manner. The actin dynamics, which comprises polymerization, branching, and depolymerization is very tightly regulated and takes place directionally at the sites of initiation of phagocytosis. Formin and profilin are two actin-binding proteins that are known to regulate actin cytoskeleton dynamics and thereby, endocytic processes. In this article, we report the participation of formin and profilin in E. histolytica phagocytosis and propose that these two proteins interact with each other and their sequential recruitment at the site is required for the successful completion of phagocytosis. The evidence is based on detailed microscopic, live imaging, interaction studies, and expression downregulation. The cells downregulated for expression of formin show absence of profilin at the site of phagocytosis, whereas downregulation of profilin does not affect formin localization.

Progressive familial intrahepatic cholestasis type 4 in an Indian child: presentation, initial course and novel compound heterozygous mutation.

A 15-year-old boy who had a history of on and off pruritus and jaundice since many years found to have a novel mutation in TJP2 gene. On examination, he had clubbing, splenomegaly, grade 3 oesophageal varices and short stature. Investigation revealed direct hyperbirubinemia with elevated liver enzymes with normal gamma-glutamyl transferase (GGT). Antinuclear antibody (ANA), smooth muscle antibody (SMA) anti-liver kidney microsomal (anti-LKM) and viral markers for hepatitis were negative. However, IgG was elevated and anti-smooth muscle antibody (ASMA) was weekly positive (1:20). He was also given a trial of steroid and azathioprine for 1 year on the basis of liver biopsy findings, raised IgG and positive ASMA but finding no improvement stopped. Genetic testing by next-generation sequencing found a novel compound heterozygous missense variation in exon 17 of the TJP2 gene confirming progressive familial intrahepatic cholestasis type 4 as the aetiology of cholestatic liver disease.

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica.

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin-dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P2 as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G-actin unlike PIPKs from other organisms. PtdIns(4,5)P2 , the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP-tagged PH-domain from PLCδ, which specifically binds PtdIns(4,5)P2 in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.

Stress-induced nuclear depletion of Entamoeba histolytica 3'-5' exoribonuclease EhRrp6 and its role in growth and erythrophagocytosis.

The 3'-5' exoribonuclease Rrp6 is a key enzyme in RNA homeostasis involved in processing and degradation of many stable RNA precursors, aberrant transcripts, and noncoding RNAs. We previously have shown that in the protozoan parasite Entamoeba histolytica, the 5'-external transcribed spacer fragment of pre-rRNA accumulates under serum starvation-induced growth stress. This fragment is a known target of degradation by Rrp6. Here, we computationally and biochemically characterized EhRrp6 and found that it contains the catalytically important EXO and HRDC domains and exhibits exoribonuclease activity with both unstructured and structured RNA substrates, which required the conserved DEDD-Y catalytic-site residues. It lacked the N-terminal PMC2NT domain for binding of the cofactor Rrp47, but could functionally complement the growth defect of a yeast rrp6 mutant. Of note, no Rrp47 homologue was detected in E. histolytica Immunolocalization studies revealed that EhRrp6 is present both in the nucleus and cytosol of normal E. histolytica cells. However, growth stress induced its complete loss from the nuclei, reversed by proteasome inhibitors. EhRrp6-depleted E. histolytica cells were severely growth restricted, and EhRrp6 overexpression protected the cells against stress, suggesting that EhRrp6 functions as a stress sensor. Importantly EhRrp6 depletion reduced erythrophagocytosis, an important virulence determinant of E. histolytica This reduction was due to a specific decrease in transcript levels of some phagocytosis-related genes (Ehcabp3 and Ehrho1), whereas expression of other genes (Ehcabp1, Ehcabp6, Ehc2pk, and Eharp2/3) was unaffected. This is the first report of the role of Rrp6 in cell growth and stress responses in a protozoan parasite.

EhRho1 regulates phagocytosis by modulating actin dynamics through EhFormin1 and EhProfilin1 in Entamoeba histolytica.

The protist parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries and a major cause of morbidity and mortality. Invasive infection in amoebiasis mostly affects intestinal epithelial cell lining but can also involve other organs, such as liver, lungs, or brain. Phagocytosis is an essential mode of nutrition in amoeba and has often been associated with virulence behaviour of E. histolytica. E. histolytica possesses a highly dynamic and actin-rich cytoskeleton that is thought to be involved in many processes, such as motility, pseudopod formation, and pathogenesis. Rho GTPases are known to be key regulators of the actin cytoskeleton and consequently influence the shape and movement of cells. Our study is mainly focused to understand the role of EhRho1 in the phagocytosis process of E. histolytica. EhRho1 got enriched in the phagocytic cups along with EhActin and remains attached with phagosomal membrane. However, there was no direct binding of EhRho1 with G- or F-actin, though binding was observed with the actin nucleating proteins EhFormin1 and EhProfilin1. Overexpression of dominant negative mutant or lowering the expression by antisense RNA of EhRho1 in trophozoites caused delocalisation of EhFormin1 and EhProfilin1 from phagocytic cups, which results in impairment of phagocytic process and decrease in F-actin content. The overall results show that EhRho1 regulates phagocytosis by modulating actin dynamics through recruitment of EhFormin1 and EhProfilin1 at the phagocytosis nucleation site in E. histolytica.

EhRho1 regulates plasma membrane blebbing through PI3 kinase in Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries. Motility of E. histolytica is important for its pathogenesis. Blebbing is an essential process contributing to cellular motility in many systems. In mammalian cells, formation of plasma membrane blebs is regulated by Rho-GTPases through its effectors, such as Rho kinase, mDia1, and acto-myosin proteins. In this study, we have illuminated the role of EhRho1 in bleb formation and motility of E. histolytica. EhRho1 was found at the site of bleb formation in plasma membrane of trophozoites. Overexpression of mutant EhRho1 defective for Guanosine triphosphate (GTP)-binding or down-regulating EhRho1 by antisense RNA resulted in reduced blebbing and motility. Moreover, serum-starvation reduced blebbing that was restored on serum-replenishment. Lysophosphatidic acid treatment induced bleb formation, whereas wortmannin inhibited the process. In all these cases, concentration of GTP-EhRho1 (active) and Phosphatidylinositol 4,5-bisphosphate (PIP2) inversely correlated with the level of plasma membrane blebbing. Our study suggests the role of EhRho1 in blebbing and bleb-based motility through PI3 kinase pathway in E. histolytica.

Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis.

Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases.