A novel variant in ADPRS disrupts ARH3 stability and subcellular localization in children with neurodegeneration and respiratory failure.

Purpose: ADP-ribosylation is a post-translational modification involving the transfer of one or more ADP-ribose units from NAD+ to target proteins. Dysregulation of ADP-ribosylation is implicated in neurodegenerative diseases. Here we report a novel homozygous variant in the ADPRS gene (c.545A>G, p.His182Arg) encoding the mono(ADP-ribosyl) hydrolase ARH3 found in 2 patients with childhood-onset neurodegeneration with stress-induced ataxia and seizures (CONDSIAS). Methods: Genetic testing via exome sequencing was used to identify the underlying disease cause in two siblings with developmental delay, seizures, progressive muscle weakness, and respiratory failure following an episodic course. Studies in a cell culture model uncover biochemical and cellular consequences of the identified genetic change. Results: The ARH3 H182R variant affects a highly conserved residue in the active site of ARH3, leading to protein instability, degradation, and reduced expression. ARH3 H182R additionally fails to localize to the nucleus. The combination of reduced expression and mislocalization of ARH3 H182R resulted in accumulation of mono-ADP ribosylated species in cells. Conclusions: The children's clinical course combined with the biochemical characterization of their genetic variant develops our understanding of the pathogenic mechanisms driving CONDSIAS and highlights a critical role for ARH3-regulated ADP ribosylation in nervous system integrity.

Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue.

Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.

A New Approach for Assessment of Neutrophil Extracellular Traps Through Immunofluorescence Staining in Whole Blood Smears.

Neutrophils, constituting 50%-70% of circulating leukocytes, play crucial roles in host defense and exhibit anti-tumorigenic properties. An elevated peripheral blood neutrophil-to-lymphocyte ratio is associated with decreased survival rates in cancer patients. In response to exposure to various antigens, neutrophils release neutrophil granular proteins, which combine to form web-like structures known as neutrophil extracellular traps (NETs). Previously, the relative percentage of NETs was found to be increased in resected tumor tissue samples from patients with gastrointestinal malignancies. The presence of NETs in peripheral blood is indicative of underlying pathological conditions. Hence, employing a non-invasive method to detect NETs in peripheral blood, along with other diagnostic tests, shows potential as a valuable tool not just for identifying different inflammatory disorders but also for assessing disease severity and determining patient suitability for surgical resection. While reliable methods exist for identifying NETs in tissue, accurately quantifying them in whole blood remains challenging. Many previous methods are time-consuming and rely on a limited set of markers that are inadequate for fully characterizing NETs. Therefore, we established a unique sensitive smear immunofluorescence assay based on blood smears to identify NETs in only as little as 2 µL of whole blood. To identify the NET complexes that have enhanced specificities, this combines the use of various antibodies against neutrophil-specific CD15, NET-specific myeloperoxidase (MPO), citrullinated histone H3 (Cit H3), and nuclear DNA. This protocol offers an easy, affordable, rapid, and non-invasive method for identifying NETs; thus, it can be utilized as a diagnostic marker and targeted through various therapeutic approaches for treating human malignancies. Key features • Characterization of neutrophil extracellular traps in whole blood smears through immunofluorescence staining. • Affordable and quantitative approach to neutrophil extracellular trap detection.

Evaluation of the Stability of Internal Fixation Constructs in a Low Distal Tibia Extra-articular Cadaveric Fracture Model: A Comparative Biomechanical Study.

Introduction Despite the recent advances in implant design, the choice of an internal fixation modality for extra-articular distal tibia fractures remains controversial, and there is sparse literature comparing the stability of intramedullary nails and locked plates for such fractures. Hence, we conducted a biomechanical study on an AO type 43A3 tibia fracture cadaveric model stabilized by four different constructs, viz., intramedullary (IM) interlocking nail, anteromedial plate, anterolateral plate, and posterior plate. An AO type 43A3 fracture is defined as an extra-articular fracture of the distal tibia with metaphyseal comminution. Methods A biomechanical comparative study on formalin-preserved human cadaveric tibiae was undertaken; a total of four groups were tested, with eight bones in each group. Out of the 32 cadaveric tibiae, 19 bones belonged to male cadavers, and 13 bones belonged to female cadavers. All bones were dissected from age-appropriate cadavers and fixed with an implant, followed by the creation of a 1 cm osteotomy to simulate an AO type 43A3 fracture. All fixation constructs were subjected to three-point bending tests in the anteroposterior (AP) and mediolateral (ML) planes. Three parameters, viz., bending stiffness, peak fracture gap angle, and neutral zone, were evaluated on the load-displacement curves. A fixation construct was deemed biomechanically stable if it had a high bending stiffness, a low neutral zone (inherent toggle in the construct by its weight), and a low peak fracture gap angle. Results Out of the four implants tested, locked IM nails exhibited the maximum biomechanical stability in terms of higher bending stiffness, smaller peak fracture gap angle, and smaller neutral zones. The IM nail exhibited the highest bending stiffness in the AP plane, and the anterolateral plate had the lowest bending stiffness, and the difference was statistically significant (p= 0.032). In the AP plane, the anterolateral plate exhibited a bending stiffness of 1.51 ± 0.69 Nm/degree, whereas the intramedullary nail exhibited a bending stiffness of 2.34 ± 0.81 Nm/degree, and the posterior locked plate had a bending stiffness of 1.57 ± 0.44 Nm/degree. In the ML plane, the anterolateral plate exhibited the highest neutral zone as compared to the intramedullary nail, which had the lowest neutral zone, and the difference was statistically significant (p = 0.019). The intramedullary nail exhibited the lowest neutral zone of 0.46 ± 0.31 degrees, whereas the posterior locked plate exhibited a neutral zone of 0.78 ± 0.43 degrees in the ML plane. The anterolateral plate exhibited a neutral zone of 1.43 ± 1.00 (expressed as mean ± SD) degrees in the mediolateral plane. Conclusion Our biomechanical study supports the recommendations of using a locked intramedullary nail for AO type 43A3 fractures. We concluded that the anterolateral plate construct exhibited the least biomechanical stability, in terms of lower AP bending stiffness and higher neutral zone. If the surgeon must choose a locked plating technique for any reason, the anterolateral locking plate should be avoided. If plating is at all required, we can recommend both anteromedial and posterior locked plating as biomechanically sound options.

Role of bio-flavonols and their derivatives in improving mitochondrial dysfunctions associated with pancreatic tumorigenesis.

Mitochondria, a cellular metabolic center, efficiently fulfill cellular energy needs and regulate crucial metabolic processes, including cellular proliferation, differentiation, apoptosis, and generation of reactive oxygen species. Alteration in the mitochondrial functions leads to metabolic imbalances and altered extracellular matrix dynamics in the host, utilized by solid tumors like pancreatic cancer (PC) to get energy benefits for fast-growing cancer cells. PC is highly heterogeneous and remains unidentified for a longer time because of its complex pathophysiology, retroperitoneal position, and lack of efficient diagnostic approaches, which is the foremost reason for accounting for the seventh leading cause of cancer-related deaths worldwide. PC cells often respond poorly to current therapeutics because of dense stromal barriers in the pancreatic tumor microenvironment, which limit the drug delivery and distribution of antitumor immune cell populations. As an alternative approach, various natural compounds like flavonoids are reported to possess potent antioxidant and anticancerous properties and are less toxic than current chemotherapeutic drugs. Therefore, we aim to summarize the current state of knowledge regarding the pharmacological properties of flavonols in PC in this review from the perspective of mitigating mitochondrial dysfunctions associated with cancer cells. Our literature survey indicates that flavonols efficiently regulate cellular metabolism by scavenging reactive oxygen species, mitigating inflammation, and arresting the cell cycle to promote apoptosis in tumor cells via intrinsic mitochondrial pathways. In particular, flavonols proficiently inhibit the cancer-associated proliferation and inflammatory pathways such as EGFR/MAPK, PI3K/Akt, and nuclear factor κB in PC. Overall, this review provides in-depth evidence about the therapeutic potential of flavonols for future anticancer strategies against PC; still, more multidisciplinary human interventional studies are required to dissect their pharmacological effect accurately.

Climate Change as a Social Determinant of Health: An Interactive Case-Based Learning Activity.

Introduction: Despite consensus on climate change's impact on humans, medical schools have not widely adopted inclusion of environmental topics into their mandatory curriculum. This educational activity explicitly addresses climate change as one of the environmental determinants of health (EDH). Methods: We developed a required, 1-hour module for all first-year medical students. This interactive, case-based, small-group activity was incorporated into a curriculum within an advising program but could be run independently. Before and after the session, participants completed evaluations assessing knowledge gains and attitude shifts. Results: Of 183 first-year students, 155 completed both pre- and postmodule surveys. Participants' rating increased on the postmodule survey item "priority should be given to the discussion of EDH in medical education." The Wilcoxon signed rank test determined this difference in priority was statistically significant (p < .001). Reported strengths of this activity included the cases, informative content on EDH, the video, the discussion, and highlighted EDH resources. Suggested areas for improvement included more information on how to apply concepts to clinical contexts, guidance on how to engage in EDH concepts, and more discussion time. As a result of the module, students planned to engage in recycling, reduced consumption, advocacy, and changes to mode of transportation. Discussion: Climate change remains the greatest global threat to human health, and future physicians must be equipped to educate patients and policymakers on the harms of environmental hazards. This brief yet effective module offers one approach to incorporating this topic into medical school curricula.

Development of B cell epitopes-based enzyme linked immune sorbent assay for detection of bovine anti-Mullerian hormone.

The present study aimed to generate antibodies against predicted B cell epitopic peptides encoding bAMH for developing different ELISA models. Sandwich ELISA was determined to be an excellent technique for assessing bAMH in bovine plasma based on sensitivity tests. The assay's specificity, sensitivity, inter- and intra-assay CV, recovery %, Lower limit of quantification (LLOQ), and Upper limit of quantification (ULOQ) were determined. The test was selective since it did not bind to AMH-related growth and differentiation factors (LH and FSH) or non-related components (BSA, progesterone). The intra-assay CV was 5.67%, 3.12%, 4.94%, 3.61% and 4.27% for 72.44, 183.11, 368.24, 522.24 and 732.25 pg/ml AMH levels, respectively. At the same time, the inter-assay CV was 8.77%, 7.87%, 4.53%, 5.76% and 6.70% for 79.30, 161.27, 356.30, 569.33 and 798.19 pg/ml AMH levels, respectively. The average (Mean ± SEM) recovery percentages were 88-100%. LLOQ was 5 pg/ml and ULOQ at 50 µg/ml (CV < 20%). In conclusion, we developed a new highly sensitive ELISA against bAMH using epitope specific antibodies.

Pre-treatment but not co-treatment with vitexin alleviates hyperthermia induced oxidative stress and inflammation in buffalo mammary epithelial cells.

This study investigated if in vitro supplementation of vitexin could mitigate the adverse effects of hyperthermia on buffalo mammary epithelial cells (BuMECs). Immortalized BuMECs were divided into seven groups (n = 3): (1) a negative control group at 37 °C; (2) BuMECs exposed to heat stress as a positive control at 42 °C for 1 h; (3-7) heat stressed BuMECs pre-treated or co-treated with different concentrations of vitexin (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM), respectively. Hyperthermia was induced by exposing the cells to 42 ºC for 1 h. For the pre-treatment experiment, BuMECs were treated with vitexin for 2 h before hyperthermia exposure. For co-treatment, vitexin was added simultaneously with hyperthermia for 1 h. Subsequently, the cells were allowed to recover for 12 h at 37 °C. Results showed that pre-treatment with vitexin was more effective than co-treatment in protecting BuMECs from hyperthermia in a dose-dependent manner, with higher concentrations (50 µM and 100 µM) being the most effective. Pre-treatment with vitexin maintained cellular viability and prevented inflammation by inducing the expression of the anti-apoptotic gene (BCL-2) and reducing the expression of the pro-apoptotic gene (Bax) and pro-inflammatory mediators (IL-1ß, IL-6) in heat-stressed BuMECs. Pre-treatment with vitexin reduced oxidative stress and induced thermotolerance by increasing the expression of antioxidants mediators such as SOD, GPx and CAT at mRNA and protein levels, and modulating the expression of heat shock proteins. The findings suggest that vitexin has the potential as a therapeutic agent to protect the mammary gland from the negative impact of hyperthermia in dairy cows.

Glycol chitosan stabilized nanomedicine of lapatinib and doxorubicin for the management of metastatic breast tumor.

Advanced breast cancer is known to be highly evasive to conventional therapeutic regimes with a 5-year survival rate of less than 30% compared to over 90% for early stages. Although several new approaches are being explored to improve the survival outcome, there is still some room for equipping existing drugs such as lapatinib (LAPA) and doxorubicin (DOX) to fight the systemic disease. LAPA is associated with poorer clinical outcomes in HER2-negative patients. However its ability to also target EGFR has warranted its use in recent clinical trials. Nevertheless, the drug is poorly absorbed post oral administration and possess low aqueous solubility. DOX on the other hand is avoided in vulnerable patients in advanced stages due to its pronounced off-target toxicity. To overcome the pitfalls of the drugs, we have fabricated a nanomedicine co-loaded with LAPA & DOX and stabilized with glycol chitosan, a biocompatible polyelectrolyte. With a loading content of ~ 11.5% and ~ 15% respectively, LAPA and DOX in a single nanomedicine showed synergistic action against triple-negative breast cancer cells in comparison to physically mixed free drugs. The nanomedicine showed a time-dependent association with cancer cells thereon inducing apoptosis leading to ~ 80% cell death. The nanomedicine was found to be acutely safe in healthy Balb/c mice and could negate DOX-induced cardio toxicity. The combination nanomedicine significantly inhibited both the primary 4T1 breast tumor and its spread to the lung, liver, heart, and kidney compared to pristine drug controls. These preliminary data indicate bright prospects for the nanomedicine to be effective against metastatic breast cancer.

Predictors of Occult Neck Metastasis and Extra Capsular Spread in Early Oral Cancers.

To investigate the incidence of occult neck metastasis and to determine the prognostic factors related to the occurrence of the cervical lymph nodal metastasis and extra capsular extension (ECE) in early oral cavity cancer patients. A retrospective review performed on 100 patients with node negative squamous cell carcinoma of oral cavity who underwent primary treatment between Jan 2015 and Dec 2018. Incidence of occult neck metastasis after the elective neck dissection in our study was 35%. Independent correlates of positive occult neck metastasis were lymphovascular Invasion (P-0.000)[CI 0.004-0.326] and depth of invasion(P-0.009)[CI 0.509-13.428] on univariate analysis and statistically significant factors associated with the incidence of the extracapsular extension were age(P-0.044), lymphovascular invasion(P-0.018)[CI 0.004-0.603] and lymph node ratio(P-0.000)[CI 4.570-158.45] on univariate analysis. Lymphovascular invasion and depth of invasion correlated significantly with occurrence of neck metastasis. Age and LVI were the prognostic factors for extra capsular spread.

Malate dehydrogenase 2 deficiency is an emerging cause of pediatric epileptic encephalopathy with a recognizable biochemical signature.

Malate dehydrogenases (MDH) serve a critical role in maintaining equilibrium of the NAD+/NADH ratio between the mitochondria and cytosol through the catalysis of the oxidation of L-malate to oxaloacetate in a reversible, NADH-dependent manner. MDH2 encodes the mitochondrial isoform, which is integral to the tricarboxylic acid cycle and thus energy homeostasis. Recently, five patients harboring compound heterozygous MDH2 variants have been described, three with early-onset epileptic encephalopathy, one with a stroke-like episode, and one with dilated cardiomyopathy. Here, we describe an additional seven patients with biallelic variants in MDH2, the largest and most neurodevelopmentally and ethnically diverse cohort to-date, including homozygous variants, a sibling pair, non-European patients, and an adult. From these patients, we learn that MDH2 deficiency results in a biochemical signature including elevations of plasma lactate and the lactate:pyruvate ratio with urinary excretion of malate. It also results in a recognizable constellation of neuroimaging findings of anterior-predominant cerebral atrophy, subependymal cysts with ventricular septations. We also recognize MDH2 deficiency as a cause of Leigh syndrome. Taken with existing patient reports, we conclude that MDH2 deficiency is an emerging and likely under-recognized cause of infantile epileptic encephalopathy and provide a framework for medical evaluation of patients identified with biallelic MDH2 variants.

Phenotype-autosomal recessive osteopetrosis.

Osteopetrosis (OPT) is a life-threatening disease characterized by increased bone mass caused by diminished osteoclast function/differentiation. The autosomal recessive forms, caused by biallelic variants in implicated genes, usually present in infancy. Without treatment, autosomal recessive OPTs are usually fatal within the first 10 years of life [1]. Here, we review the clinical features and associated pathophysiology of the autosomal recessive OPT. A greater understanding of these rare disorders will advance early diagnosis and optimal management.

Expanding the clinical spectrum of SOX18-related Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome.

Pathogenic variants in SOX18 are associated with hypotrichosis-lymphedema-telangiectasia-renal defects syndrome (HLTRS) and hypotrichosis-lymphedema-telangiectasia syndrome (HLTS). Eleven patients with SOX18 related HLTRS/HLTS have been previously described. Cardinal features include varying degrees of hypotrichosis, lymphedema and telangiectasias. We report a 15-year-old female patient with a likely de novo SOX18 pathogenic variant identified on duo exome sequencing. In addition to the classic features, the currently reported patient presented with novel clinical features including musculoskeletal abnormalities and strikingly poor wound healing. Chronic skin ulcers have been a major cause of morbidity for the patient and have led to significant functional limitation. Further, our experience with wound management has been detailed. We hope to improve understanding of the clinical spectrum of this ultra-rare disorder by reviewing the phenotypic features in all reported patients including our patient.

Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions.

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 µM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 µM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.

Ophthalmic Findings Associated with NEDD4L-related Disorder.

Pathogenic variants in the NEDD4L gene are associated with a very rare neurodevelopmental disorder characterized by periventricular nodular heterotopia, developmental delay, 2-3 toe syndactyly, and cleft palate. Ophthalmic findings associated with this disorder have not been well described in literature. We have summarized the clinical findings that have been reported in this disorder previously and highlight a novel ophthalmic finding of foveal hypoplasia in a new case of NEDD4L-related disorder.

Third reported patient with RAP1B-related syndromic thrombocytopenia and novel clinical findings.

RAP1B is a RAS-superfamily small GTP-binding protein involved in numerous cell processes. Pathogenic gain-of-function variants in this gene have been associated with RAP1B-related syndromic thrombocytopenia, an ultrarare disorder characterized by hematologic abnormalities, neurodevelopmental delays, growth delay, and congenital birth defects including cardiovascular, genitourinary, neurologic, and skeletal systems. We report a 23-year-old male with a novel, de novo RAP1B gain-of-function variant identified on genome sequencing. This is the third reported case which expands the molecular and phenotypic spectrum of RAP1B-related syndromic thrombocytopenia.

Revisiting the surgical anatomy of the triangle of doom and the triangle of pain.

Better understanding of the surgical anatomy of the triangle of doom and the triangle of pain with fixed bony landmarks like the anterior superior iliac spine (ASIS) and the pubic symphysis (PS) can help in defining a safe location for trocar placement during laparoscopic surgeries and minimize neurovascular complications. Ten cadavers were dissected bilaterally to explore the surgical anatomy of both the triangles. ASIS and PS were evaluated in relation to the deep inguinal ring, external iliac artery, femoral nerve, and inferior epigastric artery. The deep inguinal ring was located at a depth of ~3 cm, about 4.9 ± 0.56 cm along the y-axis and 6.2 ± 0.94 cm along the x-axis, from the ASIS. The external iliac artery was located ~4.33 ± 0.6 cm along the y-axis and 7.29 ± 0.76 along the x-axis from the ASIS. The inferior epigastric artery was at ~4.31 ± 0.38 cm from the midline at the level of ASIS. This knowledge can help in the surface localization of both the triangles and prevent injury to the various neurovascular structures in relation to these triangles. In the current study, cranial to the ASIS lying at a distance of >5 cm from the midline was observed to be a safe zone for accessory trocar placement. The umbilical port has been observed to be safe for trocar placement. The mean angle between ductus deferens and testicular vessels was observed to be 43.5° ± 4.79°, which could help in determining their relative locations during various surgical procedures.

MED12-related Hardikar syndrome: Two additional cases and novel phenotypic features.

Hardikar syndrome (HS) is a MED12-related ultra-rare multiple congenital malformation syndrome known to affect the gastrointestinal, cardiac, and genitourinary systems among other features including cleft lip/palate and pigmentary retinopathy. Only 10 patients affected with HS have been previously described in literature, of which seven were molecularly confirmed. We report a 20-year-old and a 13-month-old patient with HS diagnosed by exome sequencing bringing the total number of clinically diagnosed cases to 12 and MED12 associated to 9. We describe previously unreported molecular and clinical findings associated with HS and review all reported cases to permit prompt diagnosis, appropriate management, and genetic counseling of HS patients.

CCR4+ monocytic myeloid-derived suppressor cells are associated with the increased epithelial-mesenchymal transition in pancreatic adenocarcinoma patients.

Among all the cancer-related deaths globally, pancreatic ductal adenocarcinoma (PDAC) accounts for the seventh leading cause of mortality. A dysregulated immune system disrupts anti-tumor immunity by abnormal accumulation of myeloid-derived suppressor cells (MDSCs), but the underlying mechanisms are still inconclusive. To gain new insights into the role of MDSCs in tumor settings, we aimed to determine the mechanism of expansion of various subsets of MDSCs in PDAC patients and their role in promoting invasiveness. We assessed the load of MDSCs, chemokines responsible for the recruitment of MDSCs in PDAC patients by flow cytometry. We investigated the chemokine profile of tumor tissue using qRT-PCR and the status of epithelial-mesenchymal transition (EMT) related markers E-Cadherin, N-Cadherin, Snail, and ZEB1 by qRT-PCR and immunohistochemistry. We found a higher frequency of tumor infiltrated MDSCs in PDAC patients. Chemokine ligands CCL2 and the receptor CCR4 were markedly elevated in the PDAC tumor, while CCR4+ monocytic MDSCs (M-MDSCs) were found significantly elevated in peripheral blood and tumor tissue. In tumor tissue, expression of E-Cadherin was significantly reduced, while N-Cadherin, Snail, and ZEB1 were markedly raised. The frequency of CCR4+ M-MDSCs significantly correlated with the expression of mesenchymal transition markers N-Cadherin, Snail, and ZEB1. Collectively, these results suggest that the CCL2-CCR4 axis plays a crucial role in driving the recruitment of M-MDSCs, which is associated with increased invasiveness in PDAC. This study sheds light on the expansion mechanism of MDSCs, which can serve as a crucial target of future anti-cancer strategies to inhibit tumor cell invasiveness.

The anatomy of the cystic duct and its association with cholelithiasis: MR cholangiopancreatographic study.

The aims of this article are to detail the anatomy of the cystic duct in patients with and without gallstones as it relates to maneuvering of the duct during endoscopic transpapillary gallbladder cannulation, and to elucidate its role in the dynamics of bile flow during gallbladder contraction. One hundred MRCPs were retrieved from the prospectively maintained radiology data system to assess the configuration of the cystic duct and its confluence vis-a-vis the main biliary duct. The configuration of the cystic duct was broadly classified into four types: Angular (44%), Linear (40%), Spiral (11%), and Complex (5%). The level of emergence of the cystic duct from the bile duct was proximal in 29%, middle in 49% and distal in 20%. Its direction from the bile duct was to the right and angled upward in 69%, right and angled downward in 15%, left and angled upward in 13%, and left and angled downward in 1%. Its orifice was on the lateral surface of the bile duct in 50%, posterior in 19%, anterior in 15% and medial in 14%. In two cases, the cystic duct opened directly into the duodenum. Tortuous cystic ducts and non-lateral unions with the bile duct were significantly more prevalent in gallstone cases than the non-gallstone group (p = 0.02). The present study details the spatial anatomy of the cystic duct vis a vis the main biliary duct. This has not been well investigated to date but has become increasingly relevant with the advent of recent gallbladder interventions.