Single-port intragastric wedge resection using the tunnel method: a novel surgical approach for treating endophytic gastric subepithelial tumors.

BACKGROUND: Intragastric wedge resection is an effective method for treating endophytic gastric subepithelial tumors (SETs). However, retracting the stomach wall to the umbilicus is difficult in certain patients. In response, we developed a novel surgical technique for single-port intragastric wedge resection, which we termed the "tunnel method." METHODS: A transumbilical incision is made, and a wound retractor is applied. After diagnostic laparoscopy, a gastrostomy is made on the greater curvature, lower body. Another small wound retractor is inserted into the gastrostomy, and extracted through the transumbilical incision, creating a tunnel from the gastrostomy site to the umbilicus. Articulating laparoscopic instruments are inserted via the tunnel, and intragastric wedge resection is performed. We collected and analyzed the clinicopathologic and operative data of patients who underwent intragastric wedge resection via the tunnel method. RESULTS: Twenty-seven patients who underwent single-port intragastric wedge resection via the tunnel method in a single tertiary referral hospital were included in this study. The mean age of the patients was 54.6 ± 11.4 years, body mass index was 26.5 ± 3.4 kg/m2. Twenty-four (88.9%) patients had tumors located in the upper third of the stomach. The average operative time was 65.0 ± 24.2 min. None of the patients experienced Clavien-Dindo grade IIIa or higher postoperative complications. The average postoperative hospital stay length was 2.5 ± 0.8 days. Thirteen gastrointestinal stromal tumors, nine leiomyomas, and one neuroendocrine carcinoma, schwannoma, lipoma, spindle cell proliferative lesion, and fibrotic lesion were pathologically diagnosed. The average tumor size was 2.6 ± 1.3 cm. All cases had negative resection margins. CONCLUSIONS: Single-port intragastric wedge resection by the tunnel method is a feasible and safe approach for treating endophytic gastric SETs.

Effects of dry period length on milk production and physiological responses of heat-stressed dairy cows during the transition period.

The objective of this study was to investigate the effects of a traditional dry period (60 d) versus a no dry period (0 d) on the milk production, physiological response, and metabolic status of dairy cows exposed to heat stress during the transition period. Holstein dairy cows (n = 15) with similar expected calving dates were randomly assigned to two different dry period lengths: (1) no dry period (n = 7) and (2) a traditional dry period of 60 days (n = 8). All cows were studied from 8 weeks before expected calving to 10 weeks after calving and experienced heat stress during the transition period. The results showed that cows with no dry period decreased their milk yield in subsequent lactation, but compensated for the loss of milk yield accounted for by additional milk yield before calving. The energy balance at postpartum was improved in cows with no dry period compared to cows with a traditional dry period. There were no significant differences in the physiological response and blood metabolites at postpartum between the dry period lengths of dairy cows exposed to heat stress during the transition period. Taken together, our results showed that omitting the dry period improved the milk production and metabolic status of dairy cows exposed to heat stress during the transition period.

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity.

Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.

FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity.

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.

A molecular glue approach to control the half-life of CRISPR-based technologies.

Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Small Molecules for Enhancing the Precision and Safety of Genome Editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome-editing technologies have revolutionized biology, biotechnology, and medicine, and have spurred the development of new therapeutic modalities. However, there remain several barriers to the safe use of CRISPR technologies, such as unintended off-target DNA cleavages. Small molecules are important resources to solve these problems, given their facile delivery and fast action to enable temporal control of the CRISPR systems. Here, we provide a comprehensive overview of small molecules that can precisely modulate CRISPR-associated (Cas) nucleases and guide RNAs (gRNAs). We also discuss the small-molecule control of emerging genome editors (e.g., base editors) and anti-CRISPR proteins. These molecules could be used for the precise investigation of biological systems and the development of safer therapeutic modalities.

Small-molecule modulators of protein-RNA interactions.

Protein-RNA interactions (PRIs) play crucial roles in diverse cellular pathways, from transcription to liquid-liquid phase separation, and its dysregulation is associated with a wide range of human disorders. Therefore, there is great emphasis on discovering small-molecule modulators that target unexplored PRIs by developing robust PRI assays. In particular, targeting PRIs could offer innovative solutions to expand the druggable genome, as only a small portion of protein-coding genes are currently targeted by drugs. This review describes the therapeutic potential of targeting PRIs using small molecules, biochemical and cell-based experimental tools for observing PRIs, and several PRI modulators. We also highlight emerging technologies and the challenges in developing PRI modulators.

Rational Design of Silicon-Based Zinc Ionophores.

Ionophores transport ions across biological membranes and have wide-ranging applications, but a platform for their rapid development does not exist. We report a platform for developing ionophores from metal-ion chelators, which are readily available with wide-ranging affinities and specificities, and structural data that can aid rational design. Specifically, we fine-tuned the binding affinity and lipophilicity of a ZnII -chelating ligand by introducing silyl groups proximal to the ZnII -binding pocket, which generated ionophores that performed better than most of the currently known ZnII ionophores. Furthermore, these silicon-based ionophores were specific for ZnII over other metals and exhibited better antibacterial activity and less toxicity to mammalian cells than several known ZnII ionophores, including pyrithione. These studies establish rational design principles for the rapid development of potent and specific ionophores and a new class of antibacterial agents.

CRISPR-based therapeutics: current challenges and future applications.

The discovery, only a decade ago, of the genome editing power of clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases is already reinventing the therapeutic process, from how new drugs are discovered to novel ways to treat diseases. CRISPR-based screens can aid therapeutic development by quickly identifying a drug's mechanism of action and escape mutants. Additionally, CRISPR-Cas has advanced emerging ex vivo therapeutics, such as cell replacement therapies. However, Cas9 is limited as an in vivo therapeutic due to ineffective delivery, unwanted immune responses, off-target effects, unpredictable repair outcomes, and cellular stress. To address these limitations, controls that inhibit or degrade Cas9, biomolecule-Cas9 conjugates, and base editors have been developed. Herein, we discuss CRISPR-Cas systems that advance both conventional and emerging therapeutics.

Changes in milk production and blood metabolism of lactating dairy cows fed Saccharomyces cerevisiae culture fluid under heat stress.

In this study, Saccharomyces cerevisiae culture fluid (SCCF) has been added to a diet of lactating dairy cows to attempt to improve the ruminal fermentation and potentially increase the dry matter intake (DMI) and milk yield. This study was conducted to investigate the effects of SCCF on the milk yield and blood biochemistry in lactating cows during the summer. Twenty-four Holstein dairy cows were randomly assigned to one of four treatments: (1) total mixed ration (TMR-1) (Control); (2) TMR-1 supplemented with SCCF (T1); (3) TMR-2 (containing alfalfa hay) (T2); and (4) TMR-2 supplemented with SCCF (T3). SCCF (5 ml/head, 2.0×107 CFU/mL) was mixed with TMRs daily before feeding to dairy cows. The mean daily temperature-humidity index (THI) during this trial was 76.92 ± 0.51 on average and ranged from 73.04 to 81.19. For particle size distribution, TMR-2 had a lower >19 mm fraction and a higher 8-19 mm fraction than TMR-1 (p < 0.05). The type of TMR did not influence the DMI, body weight (BW), milk yield and composition, or blood metabolites. The milk yield and composition were not affected by the SCCF supplementation, but somatic cell counts were reduced by feeding SCCF (p < 0.05). Feeding SCCF significantly increased the DMI but did not affect the milk yield of dairy cows. The NEFA concentration was slightly decreased compared to that in the control and T2 groups without SCCF. Feeding a yeast culture of S. cerevisiae may improve the feed intake, milk quality and energy balance of dairy cows under heat stress.

Fluidized-Bed Granulation of Probiotics-Encapsulated Spray-Dried Skim Milk Powder: Effects of a Fluidizing Aid, Moisture-Activation and Dehydration.

A probiotic powder of poor flowability with high dust content, prepared by spray drying reconstituted skim milk fermented with Lactobacillus rhamnosus GG (LGG), was granulated by fluidized-bed granulation (FBG). The effects of the addition of skim milk powder (SMP) as a fluidizing aid, and of simple moisture-activation with or without dehydration, were investigated with respect to the performance of the FBG process. A fine, poorly fluidizable LGG powder (Geldart Group C) could be fluidized and granulated, with a 4- to 5-fold increase in particle size (d4,3 = 96-141 µm), by mixing with SMP (30-50%), which has larger, fluidizable particles belonging to Geldart Group A. Moisture-activation after the mixing, followed by fluidized-bed dehydration with hot air to remove excess moisture, further improved the FBG; the yield of the granules increased from 42% to 61% and the particle size distribution became much narrower, although the average particle size remained almost the same (d4,3 = 142 µm). These granules showed a popcorn-type structure in scanning electron microscopy images and encapsulated a sufficient level of viable LGG cells (1.6 × 108 CFU g-1). These granules also exhibited much better flowability and dispersibility than the spray-dried LGG powder.

Evaluation of heat stress responses in Holstein and Jersey cows by analyzing physiological characteristics and milk production in Korea.

We evaluated the effects of heat stress on physiological responses and milk production in Holstein and Jersey cows reared in Korea. The mean average temperature-humidity index (THI) increased significantly from May to August and then decreased until October. The mean average rectal temperature (RT) was increased in Holstein cows compared with Jersey cows, as the THI values increased from 61 to 85. The average respiratory rate (RR) was increased in Jersey cows compared with Holstein cows when the THI value increased from 61 to 85. The average surface temperature of the rumen and udder was higher in Jersey cows than in Holstein cows when the THI value increased from 61 to 85. No significant difference was noted with respect to relative serum volumes between the breeds and THI ranges, but we measured significant changes in serum pH in Holstein and Jersey cows when the THI value increased from 61 to 85. Milk production was not significantly changed in Holstein cows when the THI increased from 61 to 85, but milk production and milk protein content were significantly altered in Jersey cows when the THI increased from 61 to 85. Current study suggests that Holstein cows still have an advantage in terms of the economic returns of dairy farms in Korea. Therefore, further research is required regarding the heat tolerance of Jersey cows in Korean climatic conditions.

Effects of photoperiod and light intensity on milk production and milk composition of dairy cows in automatic milking system.

The purpose of this study was to determine the effects of photoperiod and light intensity on milk production, milk composition, hormones levels and blood metabolites indices of Korean Holstein dairy cows in automatic milking system (AMS). A total of 24 Holstein dairy cows were selected and used to four subsequent treatments for the experimental periods of 60 days. The light programs consisted of (1) Control: the natural photoperiod with 14.2 h of the light period and 9.4 h of the dark period (below 10 Lux); (2) T1: 16 h of the long day photoperiod (LDPP) with 50 Lux of light; (3) T2: 16 h of LDPP with 100 Lux of light; and (4) T3: 16 h of LDPP with 200 Lux of light, respectively. Importantly, there was a significant difference in the thurl activity of dairy cows between the different light intensity programs (p < 0.05). Milk yield was higher in T1 and T2 (40.80 ± 1.71 and 39.90 ± 2.02 kg/d, respectively) than those of Control and T3 (32.18 ± 1.51 and 35.76 ± 2.80 kg/d, respectively) (p < 0.05), but DMI was lower in T1, T2, and T3 compared to Control (p < 0.05). Also, milk fat percentage, the contents of milk fat and total solids were higher in T2 than those in the others (p < 0.05). The average daily melatonin level in milk was high to T3 (28.20 ± 0.43 pg/mL), T2 (24.62 ± 0.32 pg/mL), T1 (19.78 ± 0.35 pg/mL), and Control (19.36 ± 0.45 pg/mL) in order (p < 0.05). Also, the cortisol levels in milk and blood were lower in treatment groups than in Control (p < 0.05). The results of this study showed that it will be effective to improve the milk yield and milk composition, and to reduce the stress of dairy cows when the light conditions regulate to extend the photoperiod to 16 h at a light emitting diode (LED) intensity of 100 Lux under the AMS in dairy farm.

The effect of seasonal thermal stress on milk production and milk compositions of Korean Holstein and Jersey cows.

OBJECTIVE: In this study we investigated the effect of seasonal thermal stress on milk production and milk compositions between Holstein and Jersey dairy cows under the temperateclimate in Korea. METHODS: A total of 9 Holstein lactating dairy cows (2.0±0.11 parity) which had a daily milk yield of 29.77±0.45 kg, and days in milk of 111.2±10.29 were selected similarly at the beginning of the experiments in each season. Also, a total of 9 Jersey lactating dairy cows (1.7±0.12 parity) which had a daily milk yield of 20.01±0.43 kg, and days in milk of 114.0±9.74 were selected similarly at the beginning of the experiments. RESULTS: Results showed that the average ambient temperature (°C) and temperature-humidity index (THI) were higher in summer, and were lower in winter (p<0.05). The average relative humidity (RH, %) was higher in autumn than that of other seasons (p<0.05). Milk production was significantly decreased (Holstein 29.02 kg/d and Jersey 19.75 kg/d) in autumn than in other seasons (Holstein 30.14 kg/d and Jersey 20.96 kg/d). However, the milk production was negatively correlated in Holstein cows, and positively correlated in Jersey cows with THI values increased from 16 to 80. In addition, milk yield was increased by 15% in Holstein cows and decreased by 11% in Jersey cows with the THI values increased from 16 to 20. The fat and protein content percentage was significantly higher in Jersey milk than in Holstein milk, furthermore the fat and protein content yield was higher in Jersey cow milk than that of Holstein cow's milk with all THIs. CONCLUSION: From the study results, we concluded that Jersey cows might be less adaptable to low temperature of the winter, and this would have a negative impact on dairy farmer income since Korea's milk price estimation system places a higher value on milk yield than on milk compositions or sanitary grades.

Chemogenetic System Demonstrates That Cas9 Longevity Impacts Genome Editing Outcomes.

Prolonged Cas9 activity can hinder genome engineering as it causes off-target effects, genotoxicity, heterogeneous genome-editing outcomes, immunogenicity, and mosaicism in embryonic editing-issues which could be addressed by controlling the longevity of Cas9. Though some temporal controls of Cas9 activity have been developed, only cumbersome systems exist for modifying the lifetime. Here, we have developed a chemogenetic system that brings Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. Despite the large size of Cas9, we were able to demonstrate efficient degradation in cells from multiple species. Furthermore, by controlling the Cas9 lifetime, we were able to bias the DNA repair pathways and the genotypic outcome for both templated and nontemplated genome editing. Finally, we were able to dosably control the Cas9 activity and specificity to ameliorate the off-target effects. The ability of this system to change the Cas9 lifetime and, therefore, bias repair pathways and specificity in the desired direction allows precision control of the genome editing outcome.

Active maintenance of eligibility trace in rodent prefrontal cortex.

Even though persistent neural activity has been proposed as a mechanism for maintaining eligibility trace, direct empirical evidence for active maintenance of eligibility trace has been lacking. We recorded neuronal activity in the medial prefrontal cortex (mPFC) in rats performing a dynamic foraging task in which a choice must be remembered until its outcome on the timescale of seconds for correct credit assignment. We found that mPFC neurons maintain significant choice signals during the time period between action selection and choice outcome. We also found that neural signals for choice, outcome, and action value converge in the mPFC when choice outcome was revealed. Our results indicate that the mPFC maintains choice signals necessary for temporal credit assignment in the form of persistent neural activity in our task. They also suggest that the mPFC might update action value by combining actively maintained eligibility trace with action value and outcome signals.

Engineering designer beta cells with a CRISPR-Cas9 conjugation platform.

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a ß-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.

A comparative study on milk composition of Jersey and Holstein dairy cows during the early lactation.

Recently, Jersey cattle was introduced and produced by embryo transfer to Korea. This study was conducted to investigate the differences of milk compositions between Jersey and Holstein cows and the relationship between days in milk (DIM) and milk compositions during early lactation. Data were collected from twelve lactating cows from Department of Animal Resources Development at National Institute of Animal Science. Cows in parity 1 were used, and calved at spring from April to March of 2017. All cows were housed in two sections within a free-stall barn, which divided into six from each breed, and received a basal total mixed ration. Milk samples of each cow were collected at 3 DIM and 30 DIM for analyzing the milk compositions, including fatty acids (FA), amino acids and minerals. Total solids, citrate, and milk urea nitrogen level were differed between the breeds (p < 0.05). As DIM went from 3 to 30, milk protein, total solids, and somatic cell count decreased (p < 0.05), but lactose increased in all breed milk (p < 0.05). Citrate and free fatty acid (FFA) elevated in Jersey milk (p < 0.05), whereas reduced in Holstein milk (p < 0.05). Proportions of some individual FA varied from the breeds. Myristic (C14:0), palmitic (C16:0), and arachidonic acid (C20:4) in milk from all cows were higher at 3 DIM than at 30 DIM (p < 0.05). Also, stearic (C18:0) and oleic acid (C18:1) were lower at 3 DIM than at 30 DIM (p < 0.05), and the C18:1 to C18:0 ratio was significantly differed in DIM × breed interactions (p < 0.05). The contents of the individual amino acids did not differ from the breeds. Calcium, phosphorous, magnesium, and zinc (Zn) contents was significantly increased in Holstein milk than Jersey milk at 3 DIM. Also, K and Zn concentrations were higher in Holstein milk than in Jersey milk at 30 DIM (p < 0.05). It was concluded that Jersey cows would produce more effective milk in processing dairy products and more proper energy status compared with Holstein cows in early lactation under the same environmental and nutritional conditions.

Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay.

Protein-RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein-RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein-RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin-Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein-microRNA interactions. We clearly showed the structure-activity relationships of this new class of Lin28-let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein-RNA interactions.