Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells.

RAB11 small GTPases and associated recycling endosome have been localized to mitotic spindles and implicated in regulating mitosis. However, the physiological significance of such regulation has not been observed in mammalian tissues. We have used newly engineered mouse models to investigate intestinal epithelial renewal in the absence of single or double isoforms of RAB11 family members: Rab11a and Rab11b. Comparing with single knockouts, mice with compound ablation demonstrate a defective cell cycle entry and robust mitotic arrest followed by apoptosis, leading to a total penetrance of lethality within 3 days of gene ablation. Upon Rab11 deletion ex vivo, enteroids show abnormal mitotic spindle formation and cell death. Untargeted proteomic profiling of Rab11a and Rab11b immunoprecipitates has uncovered a shared interactome containing mitotic spindle microtubule regulators. Disrupting Rab11 alters kinesin motor KIF11 function and impairs bipolar spindle formation and cell division. These data demonstrate that RAB11A and RAB11B redundantly control mitotic spindle function and intestinal progenitor cell division, a mechanism that may be utilized to govern the homeostasis and renewal of other mammalian tissues.

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

Directional Optical Coherence Tomography Reveals Reliable Outer Nuclear Layer Measurements.

PURPOSE: Directional Optical Coherence Tomography (D-OCT) is a method used to optically segment and identify the outer nuclear layer (ONL) in vivo. The purpose of this study was to determine the repeatability and reproducibility of D-OCT ONL thickness measurements in healthy eyes. METHODS: Sixteen healthy eyes of sixteen subjects were imaged using the Cirrus SD-OCT. The OCT beam entry position was varied horizontally and vertically through the pupil, and cross-sectional images were obtained at baseline and 1-month follow-up by two observers. Detailed segmentation was performed to quantify the thickness of ONL without the inclusion of overlying Henle Fiber Layer. Inter-observer, intra-observer, and inter-visit variability was evaluated using Bland-Altman and coefficient of variation analysis for each category. RESULTS: All 16 eyes were successfully imaged, registered, and segmented. The maximum mean (SD) inter-operator difference was 2.6 (4.8) µm. The maximum mean (SD) intra-operator difference was 2.4 (5.3) µm. There was no statistically significant difference in ONL measurements detected between baseline and follow-up (p > 0.05). The mean (SD) differences measured across visits by one operator varied from -1.6 (3.1) to 1.1 (6.1) µm. The mean (SD) coefficient of variance (CV%) for all sectors with horizontal orientation was 9.1% (2.3%), 10.1% (2.5%), and 8.6% (2.3%) for inter-observer, intra-observer, and inter-visit, respectively. The mean (SD) coefficient of variance (CV%) for all sectors with vertical orientation was 8.3% (1.8%), 6.9% (1.4%), and 8.3% (2.1%) for inter-observer, intra-observer, and inter-visit, respectively. The majority of the variation of paired repeated measurements originated from between-subject variance. The within-subject variance accounted for less than 1% of the total variability. CONCLUSIONS: ONL thickness measurements can be quantified with good repeatability and reproducibility using D-OCT. Identifying the magnitude of D-OCT variability among normal subjects will allow for improved development of future clinical studies that quantitatively track the progression of macular pathology.

Dilute acid-assisted microbubbles-mediated ozonolysis of Eucheuma denticulatum phycocolloid for biobased L-lactic acid production.

Biobased L-lactic acid (L-LA) appeals to industries; however, existing technologies are plagued by limited productivity and high energy consumption. This study established an integrated process for producing macroalgae-based L-LA from Eucheuma denticulatum phycocolloid (EDP). Dilute acid-assisted microbubbles-mediated ozonolysis (DAMMO) was selected for the ozonolysis of EDP to optimize D-galactose recovery. Through single-factor optimization of DAMMO treatment, a maximum D-galactose recovery efficiency (59.10 %) was achieved using 0.15 M H2SO4 at 80 °C for 75 min. Fermentation with 3 % (w/v) mixed microbial cells (Bacillus coagulans ATCC 7050 and Lactobacillus acidophilus-14) and fermented residues achieved a 97.67 % L-LA yield. Additionally, this culture approach was further evaluated in repeated-batch fermentation and showed an average L-LA yield of 93.30 %, providing a feasible concept for macroalgae-based L-LA production.

Schizosaccharomyces pombe Hat1 (Kat1) is associated with Mis16 and is required for telomeric silencing.

The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.

Sustainable circular biorefinery approach for novel building blocks and bioenergy production from algae using microbial fuel cell.

The imminent need for transition to a circular biorefinery using microbial fuel cells (MFC), based on the valorization of renewable resources, will ameliorate the carbon footprint induced by industrialization. MFC catalyzed by bioelectrochemical process drew significant attention initially for its exceptional potential for integrated production of biochemicals and bioenergy. Nonetheless, the associated costly bioproduct production and slow microbial kinetics have constrained its commercialization. This review encompasses the potential and development of macroalgal biomass as a substrate in the MFC system for L-lactic acid (L-LA) and bioelectricity generation. Besides, an insight into the state-of-the-art technological advancement in the MFC system is also deliberated in detail. Investigations in recent years have shown that MFC developed with different anolyte enhances power density from several µW/m2 up to 8160 mW/m2. Further, this review provides a plausible picture of macroalgal-based L-LA and bioelectricity circular biorefinery in the MFC system for future research directions.

In Vitro Organoid-Based Assays Reveal SMAD4 Tumor-Suppressive Mechanisms for Serrated Colorectal Cancer Invasion.

Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4KO BRAFV600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfß pathway.

Advancement of biorefinery-derived platform chemicals from macroalgae: a perspective for bioethanol and lactic acid.

The extensive growth of energy and plastic demand has raised concerns over the depletion of fossil fuels. Moreover, the environmental conundrums worldwide integrated with global warming and improper plastic waste management have led to the development of sustainable and environmentally friendly biofuel (bioethanol) and biopolymer (lactic acid, LA) derived from biomass for fossil fuels replacement and biodegradable plastic production, respectively. However, the high production cost of bioethanol and LA had limited its industrial-scale production. This paper has comprehensively reviewed the potential and development of third-generation feedstock for bioethanol and LA production, including significant technological barriers to be overcome for potential commercialization purposes. Then, an insight into the state-of-the-art hydrolysis and fermentation technologies using macroalgae as feedstock is also deliberated in detail. Lastly, the sustainability aspect and perspective of macroalgae biomass are evaluated economically and environmentally using a developed cascading system associated with techno-economic analysis and life cycle assessment, which represent the highlights of this review paper. Furthermore, this review provides a conceivable picture of macroalgae-based bioethanol and lactic acid biorefinery and future research directions that can be served as an important guideline for scientists, policymakers, and industrial players.

Third-generation L-Lactic acid production by the microwave-assisted hydrolysis of red macroalgae Eucheuma denticulatum extract.

The development of an efficient third-generation L-lactic acid (L-LA) production process from Eucheuma denticulatum extract (EDE) was achieved in this study. Microwave-assisted dilute acid hydrolysis (MADAH) and microwave-assisted hydrothermal hydrolysis (MAHTH) were chosen as the hydrolysis of EDE for the objective of increasing galactose yield. Single-factor optimization of hydrolysis of the EDE was studied, MADAH had high performance in galactose production relative to MAHTH, in which the yield and optimal conditions for both processes were 50.7% (0.1 M H2SO4, 120 °C for 25 min) and 47.8% (0 M H2SO4,160 °C for 35 min), respectively. For fermentation, the optimal L-LA yield was achieved at the inoculum cell density of 4% (w/w) Bacillus coagulans ATCC 7050 with 89.4% and 6% (w/w) Lactobacillus acidophilus LA-14 with 87.6%. In addition, lipid-extracted Chlorella vulgaris residues (CVRs) as co-nutrient supplementation increased the relative abundance of B. coagulans ATCC 7050, thus benefiting L-LA production.

Sustainable and green pretreatment strategy of Eucheuma denticulatum residues for third-generation l-lactic acid production.

Managing plastic waste remains an urgent environmental concern and switching to biodegradable plastics can reduce the dependence on depleting fossil fuels. This study emphasises the efficacy of macroalgae wastes, Eucheuma denticulatum residues (EDRs), as potential alternate feedstock to produce l-lactic acid (l-LA), the monomer of polylactic acid, through fermentation. An innovative environmental friendly strategy was explored in this study to develop a glucose platform from EDRs: pretreatment with microwave-assisted autohydrolysis (MAA) applied to enhance enzymatic hydrolysis of EDRs. The results indicate that MAA pretreatment significantly increased the digestibility of EDRs during the enzymatic hydrolysis process. The optimum pretreatment conditions were 120 °C and 50 min, resulting in 96.5% of enzymatic digestibility after 48 h. The high l-LA yield of 98.6% was obtained using pretreated EDRs and supplemented with yeast extract. The energy analysis implies that MAA pretreatment could further improve the overall energy efficiency of the process.

SOX4 and SMARCA4 cooperatively regulate PI3k signaling through transcriptional activation of TGFBR2.

Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.

SMAD4 is critical in suppression of BRAF-V600E serrated tumorigenesis.

BRAF-driven colorectal cancer is among the poorest prognosis subtypes of colon cancer. Previous studies suggest that BRAF-mutant serrated cancers frequently exhibit Microsatellite Instability (MSI) and elevated levels of WNT signaling. The loss of tumor-suppressor Smad4 in oncogenic BRAF-V600E mouse models promotes rapid serrated tumor development and progression, and SMAD4 mutations co-occur in human patient tumors with BRAF-V600E mutations. This study assesses the role of SMAD4 in early-stage serrated tumorigenesis. SMAD4 loss promotes microsatellite stable (MSS) serrated tumors in an oncogenic BRAF-V600E context, providing a model for MSS serrated cancers. Inactivation of Msh2 in these mice accelerated tumor formation, and whole-exome sequencing of both MSS and MSI serrated tumors derived from these mouse models revealed that all serrated tumors developed oncogenic WNT mutations, predominantly in the WNT-effector gene Ctnnb1 (ß-catenin). Mouse models mimicking the oncogenic ß-catenin mutation show that the combination of three oncogenic mutations (Ctnnb1, Braf, and Smad4) are critical to drive rapid serrated dysplasia formation. Re-analysis of human tumor data reveals BRAF-V600E mutations co-occur with oncogenic mutations in both WNT and SMAD4/TGFß pathways. These findings identify SMAD4 as a critical factor in early-stage serrated cancers and helps broaden the knowledge of this rare but aggressive subset of colorectal cancer.

Elevating EGFR-MAPK program by a nonconventional Cdc42 enhances intestinal epithelial survival and regeneration.

The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal stem cells (ISCs) strictly required growth response mediated by cell division control 42 (Cdc42) and Cdc42-deficient enteroids to undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated, receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiologically, but nonconventionally, spliced Cdc42 variant 2 (V2) exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration, and elevating this signaling cascade is capable of initiating protection from genotoxic injury.

Is the Osmolal Concentration of Ethanol Greater Than Its Molar Concentration?

Background: Recent data suggested that the osmolal gap attributed to ethanol as determined by the difference between measured serum osmolality and calculated serum osmolarity is greater than its molar concentration. The increased osmotic activity of ethanol is thought to be due to its binding to water molecules. This study is conducted to determine the true osmotic contribution of ethanol to serum osmolality. Methods: Baseline serum osmolality and ethanol concentration were measured on each serum sample. Varying amounts of ethanol were added to aliquots of serum in which the baseline serum ethanol concentration was undetectable. Repeat serum osmolality and serum ethanol concentration were measured after addition of ethanol. Results: The range of serum ethanol concentration was 27.3-429.8 mg/dL. The serum osmolal gap attributed solely to ethanol is calculated based on the difference between measured serum osmolality before and measured serum osmolality after addition of ethanol. Our results demonstrated that the contribution of ethanol to serum osmolality can be calculated by dividing the serum ethanol level in mg/dl by 4.6. In addition, the relationship between serum ethanol concentration and osmolal gap due to ethanol was assessed by linear regression analysis. Linear regression analysis relating the osmolal gap due to ethanol and ethanol concentration yielded the following equation: Osmolal Gap (mOsm/kg H2O) = 0.23 (Ethanol [mg/dL]) - 1.43. Conclusion: The osmolal concentration of ethanol can be calculated based on its molar concentration. We found no evidence for ethanol binding to water molecules over the range of ethanol concentration in this study.

Recycling Endosomes in Mature Epithelia Restrain Tumorigenic Signaling.

The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. SIGNIFICANCE: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism.

Paneth Cell Multipotency Induced by Notch Activation following Injury.

Paneth cells are post-mitotic intestinal epithelial cells supporting the stem cell niche and mucosal immunity. Paneth cell pathologies are observed in various gastrointestinal diseases, but their plasticity and response to genomic and environmental challenges remain unclear. Using a knockin allele engineered at the mouse Lyz1 locus, we performed detailed Paneth cell-lineage tracing. Irradiation induced a subset of Paneth cells to proliferate and differentiate into villus epithelial cells. RNA sequencing (RNA-seq) revealed that Paneth cells sorted from irradiated mice acquired a stem cell-like transcriptome; when cultured in vitro, these individual Paneth cells formed organoids. Irradiation activated Notch signaling, and forced expression of Notch intracellular domain (NICD) in Paneth cells, but not Wnt/ß-catenin pathway activation, induced their dedifferentiation. This study documents Paneth cell plasticity, particularly their ability to participate in epithelial replenishment following stem cell loss, adding to a growing body of knowledge detailing the molecular pathways controlling injury-induced regeneration.

Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer.

Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.

Cohesin without cohesion: a novel role for Pds5 in Saccharomyces cerevisiae.

High fidelity chromosome segregation during mitosis requires that cells identify the products of DNA replication during S-phase and then maintain that identity until anaphase onset. Sister chromatid identity is achieved through cohesin complexes (Smc1, Smc3, and Mcd1 and Irr1/Scc3), but the structure through which cohesins perform this task remains enigmatic. In the absence of unambiguous data, a popular model is that a subset of cohesin subunits form a huge ring-like structure that embraces both sister chromatids. This 'one-ring two-sister chromatid embrace' model makes clear predictions--including that premature cohesion loss in mitotic cells must occur through a substantial reduction in cohesin-DNA associations. We used chromatin immunoprecipitation to directly test for cohesin dissociation from well-established cohesin binding sites in mitotic cells inactivated for Pds5--a key cohesin regulatory protein. The results reveal little if any chromatin dissociation from cohesins, despite a regimen that produces both massive loss of sister chromatid tethering and cell inviability. We further excluded models that cohesion loss in mitotic cells inactivated for Pds5 arises through either cohesin subunit degradation, premature Hos1-dependent Smc3 de-acetylation or Rad61/WAPL-dependent regulation of cohesin dynamics. In combination, our findings support a model that cohesin complexes associate with each sister and that sister chromatid cohesion likely results from cohesin-cohesin interactions. We further assessed the role that Pds5 plays in cohesion establishment during S-phase. The results show that Pds5 inactivation can result in establishment defects despite normal cohesion loading and Smc3 acetylation, revealing a novel establishment role for Pds5 that is independent of these processes. The combination of findings provides important new insights that significantly impact current models of both cohesion establishment reactions and maintenance.