Larval morphology of Belgica antarctica Jacobs, 1900 (Diptera, Chironomidae, Orthocladiinae) from central part of the maritime Antarctic and deformities found in the larvae.

The larval morphology of the endemic species Belgica antarctica Jacobs, collected in January and February (2022) from six sites of Antarctica, was studied. The mouth apparatus and the parapods of one hundred seventy-six larvae were analyzed. No differences were found in the morphology of these structures between individuals of different sites. However, differences (with the exception of the length of 2nd antennal segment) were estimated in the sizes of the morphological features, as 2023 measurements were made of fifteen kinds of these structures and larval body. Deformities of mentum, mandible, antennal blade and antennal segments in percentage were established in larvae of almost all of the studied sites. The reasons for the differences and deformities are discussed.

Belgica antarctica (Diptera: Chironomidae): A natural model organism for extreme environments.

Belgica antarctica (Diptera: Chironomidae), a brachypterous midge endemic to the maritime Antarctic, was first described in 1900. Over more than a century of study, a vast amount of information has been compiled on the species (3 750 000 Google search results as of January 10, 2021), encompassing its ecology and biology, life cycle and reproduction, polytene chromosomes, physiology, biochemistry and, increasingly, omics. In 2014, B. antarctica's genome was sequenced, further boosting research. Certain developmental stages can be cultured successfully in the laboratory. Taken together, this wealth of information allows the species to be viewed as a natural model organism for studies of adaptation and function in extreme environments.

External Morphology of Larvae of Belgica antarctica Jacobs, 1900 (Diptera, Chironomidae) Obtained from Two Locations in Maritime Antarctica.

The external morphology of the fourth-instar larva of the Antarctic endemic chironomid midge Belgica antarctica is described. Larvae were collected from Jougla Point (Wiencke Island) and an un-named island close to Enterprise Island, off the coast of the western Antarctic Peninsula. Light microscopy was used to examine and document photographically the structures of the mouthparts (mandible, mentum, premandible, labrum), antennae, pecten epipharyngis, clypeus, frontal apotome and posterior parapods. Measurements of the mouthparts are presented. The data obtained are compared with that available in the literature. A number of differences were identified relating to the size of the larvae, the number of teeth on the mandibles, the number of antennal segments and the length of the antennal blade. Malformations of the mandible and mentum are reported for the first time in this species. Features of larvae of taxonomic value that can be used to determine the species in larval stages are presented. These are of utility in using the larvae to reveal relationships with other species. Larvae are also important in ecological and genotoxicological studies, which require accurate species level identification.

Vitamin D Signaling Suppresses Early Prostate Carcinogenesis in TgAPT121 Mice.

We tested whether lifelong modification of vitamin D signaling can alter the progression of early prostate carcinogenesis in studies using mice that develop high-grade prostatic intraepithelial neoplasia that is similar to humans. Two tissue-limited models showed that prostate vitamin D receptor (VDR) loss increased prostate carcinogenesis. In another study, we fed diets with three vitamin D3 levels (inadequate = 25 IU/kg diet, adequate for bone health = 150 IU/kg, or high = 1,000 IU/kg) and two calcium levels (adequate for bone health = 0.5% and high = 1.5%). Dietary vitamin D caused a dose-dependent increase in serum 25-hydroxyvitamin D levels and a reduction in the percentage of mice with adenocarcinoma but did not improve bone mass. In contrast, high calcium suppressed serum 1,25-dihydroxyvitamin D levels and improved bone mass but increased the incidence of adenocarcinoma. Analysis of the VDR cistrome in RWPE1 prostate epithelial cells revealed vitamin D-mediated regulation of multiple cancer-relevant pathways. Our data support the hypothesis that the loss of vitamin D signaling accelerates the early stages of prostate carcinogenesis, and our results suggest that different dietary requirements may be needed to support prostate health or maximize bone mass. SIGNIFICANCE: This work shows that disrupting vitamin D signaling through diet or genetic deletion increases early prostate carcinogenesis through multiple pathways. Higher-diet vitamin D levels are needed for cancer than bone.

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation.

BACKGROUND/AIMS: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. METHODS: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. RESULTS: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. CONCLUSIONS: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

Schlafen 12 expression modulates prostate cancer cell differentiation.

BACKGROUND: Schlafen proteins have previously been linked to leukocyte and intestinal epithelial differentiation. We hypothesized that Schlafen 12 (SLFN12) overexpression in human prostate epithelial cells would modulate expression of prostate-specific antigen (PSA) and dipeptidyl peptidase 4 (DPP4), markers of prostatic epithelial differentiation. MATERIALS AND METHODS: Differentiation of the human prostate cancer cell lines LNCaP and PC-3 was compared after infection with an adenoviral vector coding for SLFN12 (Ad-SLFN12) or green fluorescent protein (GFP) only expressing virus (control). Transcript levels of SLFN12, PSA, and DPP4 were evaluated by real-time reverse transcription PCR and protein levels by Western blotting. Because mixed lineage kinase (MLK) and one of its downstream effectors (extracellular signal-regulated kinases [ERK]) have previously been implicated in some aspects of prostate epithelial differentiation, we conducted further studies in which LNCaP cells were cotreated with dimethyl sulfoxide (control), PD98059 (ERK inhibitor), or MLK inhibitor during transfection with Ad-SLFN12 for 72 h. RESULTS: Treatment of LNCaP or PC-3 cells with Ad-SLFN12 reduced PSA expression by 56.6±4.6% (P<0.05) but increased DPP4 transcript level by 4.8±1.0 fold (P<0.05) versus Ad-GFP-treated controls. Further studies in LNCaP cells showed that Ad-SLFN12 overexpression increased the ratio of the mature E-cadherin protein to its precursor protein. Furthermore, SLFN12 overexpression promoted DPP4 expression either when MLK or ERK was blocked. ERK inhibition did not reverse SLFN12-induced changes in PSA, E-cadherin, or DPP4. CONCLUSIONS: SLFN12 may regulate differentiation in prostate epithelial cells, at least in part independently of ERK or MLK. Understanding how SLFN12 influences prostatic epithelial differentiation may ultimately identify targets to influence the phenotype of prostatic malignancy.

Regulation of epithelial differentiation in rat intestine by intraluminal delivery of an adenoviral vector or silencing RNA coding for Schlafen 3.

Although we stimulate enterocytic proliferation to ameliorate short gut syndrome or mucosal atrophy, less effort has been directed at enterocytic differentiation. Schlafen 3 (Slfn3) is a poorly understood protein induced during IEC-6 enterocytic differentiation. We hypothesized that exogenous manipulation of Slfn3 would regulate enterocytic differentiation in vivo. Adenoviral vector coding for Slfn3 cDNA (Ad-GFP-Slfn3) or silencing RNA for Slfn3 (siSlfn3) was introduced intraluminally into rat intestine. We assessed Slfn3, villin, sucrase-isomaltase (SI), Dpp4, and Glut2 by qRT-PCR, Western blot, and immunohistochemistry. We also studied Slfn3 and these differentiation markers in atrophic defunctionalized jejunal mucosa and the crypt-villus axis of normal jejunum. Ad-GFP-Slfn3 but not Ad-GFP increased Slfn3, villin and Dpp4 expression in human Caco-2 intestinal epithelial cells. Injecting Ad-GFP-Slfn3 into rat jejunum in vivo increased mucosal Slfn3 mRNA three days later vs. intraluminal Ad-GFP. This Slfn3 overexpression was associated with increases in all four differentiation markers. Injecting siSlfn3 into rat jejunum in vivo substantially reduced Slfn3 and all four intestinal mucosal differentiation markers three days later, as well as Dpp4 specific activity. Endogenous Slfn3 was reduced in atrophic mucosa from a blind-end Roux-en-Y anastomosis in parallel with differentiation marker expression together with AKT and p38 signaling. Slfn3 was more highly expressed in the villi than the crypts, paralleling Glut2, SI and Dpp4. Slfn3 is a key intracellular regulator of rat enterocytic differentiation. Understanding how Slfn3 works may identify targets to promote enterocytic differentiation and maintain mucosal function in vivo, facilitating enteral nutrition and improving survival in patients with mucosal atrophy or short gut syndrome.

The correlation between the expression of differentiation markers in rat small intestinal mucosa and the transcript levels of schlafen 3.

IMPORTANCE: The normal absorptive function and structural maintenance of the intestinal mucosa depend on a constant process of proliferation of enterocytic stem cells followed by progressive differentiation toward a mature phenotype. The mechanisms that govern enterocytic differentiation in the mucosa of the small intestine are poorly understood. OBJECTIVE: To determine whether schlafen 3 (but not other schlafen proteins) act in vivo and whether its effects are limited to the small intestine. We have previously demonstrated in nonmalignant rat intestinal IEC-6 cells that schlafen 3 levels correlate with the expression of various differentiation markers in vitro in response to differentiation stimuli. DESIGN: Randomized controlled experiment. SETTING: Animal science laboratory. PARTICIPANTS: Male Sprague-Dawley rats 8 to 13 weeks old. MAIN OUTCOMES AND MEASURES: Messenger RNA (mRNA) from jejunal and colonic mucosa was isolated, and transcript levels of schlafen proteins 1, 2, 3, 4, 5, 13, and 14; sucrase isomaltase (SI); dipeptidyl peptidase 4 (Dpp4); glucose transporter type 2 (Glut2); and villin were measured by quantitative reverse transcriptase-polymerase chain reaction. We tested parallel variations in protein levels by Western blotting and Dpp4 enzyme activity. RESULTS: The transcript level of schlafen 3 (Slfn3) correlated with the levels of the differentiation markers SI, Dpp4, Glut2, and villin. However, the expression of schlafen proteins 1, 2, 4, 5, 13, and 14 did not correlate with the expression of the differentiation markers. The mucosal mRNA levels of Slfn3, SI, Glut2, and Dpp4 were all substantially higher in the rat jejunum than in colonic mucosa by a mean (SE) factor of 51.0 (13.2) for 6 rats (P < .05), 599 (99) for 8 rats (P < .01), 12.5 (5.5) for 8 rats (P < .01), and 14.0 (3.9) for 8 rats (P < .01), respectively. In IEC-6 cells, infection with adenovirus-expressing GFP-tagged Slfn3 significantly increased Slfn3 expression and Dpp4-specific activity compared with GFP-expressing virus (in 6 rats; P < .05). CONCLUSIONS AND RELEVANCE: Taken together with our previous in vitro observations, the results suggest that small intestinal enterocytic epithelial differentiation in rats may be regulated by Slfn3 in vivo, as in vitro, and that these effects may be specific to the small intestinal enterocytic phenotype as opposed to that of the mature colonocyte. Slfn3 human orthologs may be targeted to stimulate intestinal differentiation in patients with short bowel syndrome.

Influence of defunctionalization and mechanical forces on intestinal epithelial wound healing.

The influence on mucosal healing of luminal nutrient flow and the forces it creates are poorly understood. We hypothesized that altered deformation and extracellular pressure mediate, in part, the effects of defunctionalization on mucosal healing. We created patent or partially obstructing defunctionalizing jejunal Roux-en-Y anastomoses in rats to investigate mucosal healing in the absence or presence of luminal nutrient flow and measured luminal pressures to document partial obstruction. We used serosal acetic acid to induce ulcers in the proximal, distal, and defunctionalized intestinal segments. After 3 days, we assessed ulcer area, proliferation, and phosphorylated ERK. In vitro, we measured proliferation and migration in Caco-2 and IEC-6 intestinal epithelial cells subjected to cyclic strain, increased extracellular pressure, or strain and pressure together. Defunctionalization of intestine without obstruction reduced phosphorylated ERK, slowed ulcer healing, and inhibited mucosal proliferation. This outcome was blocked by PD-98059. Partial obstruction delayed ulcer healing but stimulated proliferation independently of ERK. In vitro, strain increased Caco-2 and IEC-6 proliferation and reduced migration across collagen but reduced proliferation and increased migration across fibronectin. In contrast, increased pressure and the combination of pressure and strain increased proliferation and reduced migration independently of substrate. PD-98059 reduced basal migration but increased migration under pressure. These results suggest that loss of the repetitive distension may decrease mucosal healing in defunctionalized bowel, while increased luminal pressure above anastomoses or in spastic bowel disease could further inhibit mucosal healing, despite peristaltic repetitive strain. ERK may mediate the effects of repetitive deformation but not the effects of pressure.

Loss of MLK3 signaling impedes ulcer healing by modulating MAPK signaling in mouse intestinal mucosa.

Mixed-lineage kinase 3 (MLK3) activates multiple MAPK pathways and can initiate apoptosis, proliferation, migration, or differentiation in different cell types. However, whether MLK3 signaling regulates intestinal epithelial cell sheet migration in vivo is not known. We sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility in vivo and in vitro. In vivo, we compared the healing of jejunal mucosal ulcers induced in MLK3 knockout (KO) mice with healing in wild-type (WT) mice. Ulcer healing was 20.8% less at day 3 (P < 0.05) and 18.9% less at day 5 (P < 0.05) in MLK3 KO than WT mice. Within the intestinal mucosa of MLK3 KO mice, ERK and JNK signaling were reduced, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) level was increased, and p38 signaling was unchanged. Parallel in vitro studies using an MLK inhibitor assessed the role of MLK signaling in human Caco-2 intestinal epithelial migration across collagen substrates. The MLK inhibitor reduced closure of circular wounds in Caco-2 monolayers. MLK inhibition reduced ERK and JNK, but not p38, signaling in Caco-2 cells. Although PTEN is increased after MLK inhibition, it does not influence MLK-mediated cell migration. These findings indicate that disruption of MLK3 signaling impairs ulcer healing by suppressing ERK and JNK signaling in vitro and in mouse intestinal mucosa in vivo. These results reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration in vivo and suggest that MLK3 may be an important target for the regulation of intestinal mucosal healing.

Changes in morphology and function in small intestinal mucosa after Roux-en-Y surgery in a rat model.

BACKGROUND: Currently there is no an appropriate model to study intestinal mucosal atrophy in vivo that preserves the nutritional status of the organism. MATERIALS AND METHODS: We created a defunctionalized segment of jejunum via a dead-end Roux-en-Y anastomosis in rats. We compared tissue morphometric parameters in the intestinal mucosa of the defunctionalized bowel with that of the mucosa proximal and distal to the anastomosis. We further measured extracellular signal-regulated kinase (ERK) activation within the mucosa as well as sucrase-isomaltase and dipeptidyl peptidase-4 levels as markers of intestinal mucosal differentiation by Western blotting of mucosal scrapings. RESULTS: Three days after anastomosis, the defunctionalized bowel exhibited decreased diameter and thickness of both the mucosa and the fibromuscular layer compared with adjacent bowel in continuity for luminal nutrient flow or with bowel from control animals. Sucrase-isomaltase and dipeptidyl peptidase-4 levels also were decreased. Furthermore, mucosal ERK activation, assessed as the ratio of phosphorylated to total ERK, also was reduced. Animal weights did not differ between bypassed and control animals. CONCLUSIONS: Deprivation of nutrient flow in a segment of bowel by defunctionalizing Roux-en-anastomosis produces mucosal atrophy as indicated by altered histology, differentiation marker expression, and ERK signaling, in animals that are otherwise able to maintain enteral nutrition.

Dietary vitamin D and vitamin D receptor level modulate epithelial cell proliferation and apoptosis in the prostate.

Low vitamin D (VD) status may increase prostate cancer risk but experimental evidence for this relationship is modest. We tested whether low VD status or VD receptor (VDR) deletion influences prostate epithelial cell (PEC) biology using intact mice, castrated mice, or castrated mice treated with testosterone propionate (TP, 2.5 mg/kg BW). PEC proliferation (Ki-67 staining) and apoptosis (TUNEL method) were determined in the anterior prostate (AP). In study 1, wild-type (WT) and TgAPT(121) mice (a model of prostate intraepithelial neoplasia) were fed diets with 25, 200 (reference diet), or 10,000 IU VD/kg diet (as vitamin D(3)) prior to castration/repletion. Serum 25 hydroxyvitamin D levels were 26, 78, and 237 nmol/L in the three diet groups, respectively. Castration reduced proliferation and increased apoptosis in the AP while TP reversed these effects. Low VD diet increased proliferation in WT (+82%) and TgAPT(121) (+24%) mice while it suppressed apoptosis in WT (-29%) and TgAPT(121) (-37%) mice. This diet also increased the severity of prostate intraepithelial neoplastic lesions in the AP of intact TgAPT(121) mice. In study 2, mice with PEC-specific VDR deletion (PEC VDR KO) were examined after castration/repletion. TUNEL staining was 60% lower in castrated PEC VDR KO mice compared with castrated WT mice. In castrated mice given TP, Ki-67 staining was 2-fold higher in PEC VDR KO compared with WT mice. Our data show that low diet VDR or VDR deletion provide a prostate environment that is permissive to early procarcinogenic events that enhance prostate cancer risk.

Constitutive activation of the mitogen-activated protein kinase pathway impairs vitamin D signaling in human prostate epithelial cells.

We studied the effect of prolonged activation of mitogen-activated protein kinase (MAPK) signaling on 1,25 dihydroxyvitamin D (1,25(OH)(2)D(3)) action in the immortalized human prostate epithelial cell line RWPE1 and its Ki-Ras transformed clone RWPE2. 1,25(OH)(2)D(3)-treatment caused growth arrest and induced gene expression in both cell lines but the response was blunted in RWPE2 cells. Vitamin D receptor (VDR) levels were lower in RWPE2 cells but VDR over-expression did not increase vitamin-D-mediated gene transcription in either cell line. In contrast, MAPK inhibition restored normal vitamin D transcriptional responses in RWPE2 cells and MAPK activation with constitutively active MEK1R4F reduced vitamin-D-regulated transcription in RWPE1 cells. 1,25(OH)(2)D(3)-mediated transcription depends upon the VDR and its heterodimeric partner the retinoid X receptor (RXR) so we studied whether changes in the VDR-RXR transcription complex occur in response to MAPK activation. Mutation of putative phosphorylation sites in the activation function 1 (AF-1) domain (S32A, T82A) of RXRalpha restored 1,25(OH)(2)D(3)-mediated transactivation in RWPE2 cells. Mammalian two-hybrid and co-immunoprecipitation assays revealed a vitamin-D-independent interaction between steroid receptor co-activator-1 (SRC-1) and RXRalpha that was reduced by MAPK activation and was restored in RWPE2 cells by mutating S32 and T82 in the RXRalpha AF-1 domain. Our data show that a common contributor to cancer development, prolonged activation of MAPK signaling, impairs 1,25(OH)(2)D(3)-mediated transcription in prostate epithelial cells. This is due in part to the phosphorylation of critical amino acids in the RXRalpha AF-1 domain and impaired co-activator recruitment.

1,25 dihydroxyvitamin D-mediated orchestration of anticancer, transcript-level effects in the immortalized, non-transformed prostate epithelial cell line, RWPE1.

BACKGROUND: Prostate cancer is the second leading cause of cancer mortality among US men. Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1alpha,25 dihydroxyvitamin D3 (1,25(OH)2D) has anti-cancer effects in cultured prostate cells. Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown. RESULTS: We examined the effect of 1,25(OH)2D (+/- 100 nM, 6, 24, 48 h) on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH)2D (Affymetrix U133 Plus 2.0, n = 4/treatment per time and dose). Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60% up), 48 h, 3566 (38% up). 288 transcripts were regulated similarly at all time points (182 up, 80 down) and many of the promoters for these transcripts contained putative vitamin D response elements. Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling. Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways). There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h. CONCLUSIONS: Our data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention. In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis.