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Objective: The number of individuals affected by metabolic dysfunction associated fatty liver disease [1] is on the rise, yet hormonal contributors to the condition remain incompletely described and only a single FDA-approved treatment is available. Some studies suggest that the hormones ghrelin and LEAP2, which act as agonist and antagonist/inverse agonist, respectively, for the G protein coupled receptor GHSR, may influence the development of MAFLD. For instance, ghrelin increases hepatic fat whereas synthetic GHSR antagonists do the opposite. Also, hepatic steatosis is less prominent in standard chow-fed ghrelin-KO mice but more prominent in 42% high-fat diet-fed female LEAP2-KO mice. Methods: Here, we sought to determine the therapeutic potential of a long-acting LEAP2 analog (LA-LEAP2) to treat MAFLD in mice. LEAP2-KO and wild-type littermate mice were fed a Gubra-Amylin-NASH (GAN) diet for 10 or 40 wks, with some randomized to an additional 28 or 10 days of GAN diet, respectively, while treated with LA-LEAP2 vs Vehicle. Various metabolic parameters were followed and biochemical and histological assessments of MAFLD were made. Results: Among the most notable metabolic effects, daily LA-LEAP2 administration to both LEAP2-KO and wild-type littermates during the final 4 wks of a 14 wk-long GAN diet challenge markedly reduced liver weight, hepatic triglycerides, plasma ALT, hepatic microvesicular steatosis, hepatic lobular inflammation, NASH activity scores, and prevalence of higher-grade fibrosis. These changes were accompanied by prominent reductions in body weight, without effects on food intake, and reduced plasma total cholesterol. Daily LA-LEAP2 administration during the final 10 d of a 41.5 wk-long GAN diet challenge also reduced body weight, plasma ALT, and plasma total cholesterol in LEAP2-KO and wild-type littermates and prevalence of higher grade fibrosis in LEAP2-KO mice. Conclusions: Administration of LA-LEAP2 to mice fed a MAFLD-prone diet markedly improves several facets of MAFLD, including hepatic steatosis, hepatic lobular inflammation, higher-grade hepatic fibrosis, and transaminitis. These changes are accompanied by prominent reductions in body weight and lowered plasma total cholesterol. Taken together, these data suggest that LEAP2 analogs such as LA-LEAP2 hold promise for the treatment of MAFLD and obesity.

Abstract Background Ewing sarcoma (EWS) is a malignancy which primarily arises in adolescence and has been studied extensively in this population. Much less is known about the rare patient cohort over the age of 40 at diagnosis. In this study, we describe the survival outcomes and clinical characteristics of this population. Methods This retrospective cohort study utilized the National Cancer Database (NCDB) to identify 4600 patients diagnosed between 2004 through 2019. Of these patients, 4058 were under the age of 40 and 542 were over 40. Propensity score 1:1 matching was performed according to sex and race. Univariate and multivariate logistic regression was performed to generate odds ratios (OR) and a Multivariate Cox regression model was used to generate a hazard ratio (HR) for patients over 40. Kaplan–Meier curves were used to estimate survival from diagnosis to death between age groups. Chi-square tests were used to compare demographic and socioeconomic patient characteristics. IBM statistics version 27.0 was used. p

Abstract Norepinephrine (NE) is a well-known appetite regulator, and the nor/adrenergic system is targeted by several anti-obesity drugs. To better understand the circuitry underlying adrenergic appetite control, here we investigated the paraventricular hypothalamic nucleus (PVN), a key brain region that integrates energy signals and receives dense nor/adrenergic input, using a mouse model. We found that PVN NE level increases with signals of energy deficit and decreases with food access. This pattern is recapitulated by the innervating catecholaminergic axon terminals originating from NTSTH-neurons. Optogenetic activation of rostral-NTSTH → PVN projection elicited strong motivation to eat comparable to overnight fasting whereas its inhibition attenuated both fasting-induced & hypoglycemic feeding. We found that NTSTH-axons functionally targeted PVNMC4R-neurons by predominantly inhibiting them, in part, through α1-AR mediated potentiation of GABA release from ARCAgRP presynaptic terminals. Furthermore, glucoprivation suppressed PVNMC4R activity, which was required for hypoglycemic feeding response. These results define an ascending nor/adrenergic circuit, NTSTH → PVNMC4R, that conveys peripheral hunger signals to melanocortin pathway.

Summary: Opioids are generally known to promote hedonic food consumption. Although much of the existing evidence is primarily based on studies of the mesolimbic pathway, endogenous opioids and their receptors are widely expressed in hypothalamic appetite circuits as well; however, their role in homeostatic feeding remains unclear. Using a fluorescent opioid sensor, deltaLight, here we report that mediobasal hypothalamic opioid levels increase by feeding, which directly and indirectly inhibits agouti-related protein (AgRP)-expressing neurons through the μ-opioid receptor (MOR). AgRP-specific MOR expression increases by energy surfeit and contributes to opioid-induced suppression of appetite. Conversely, its antagonists diminish suppression of AgRP neuron activity by food and satiety hormones. Mice with AgRP neuron-specific ablation of MOR expression have increased fat preference without increased motivation. These results suggest that post-ingestion release of endogenous opioids contributes to AgRP neuron inhibition to shape food choice through MOR signaling.

Neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires transendothelial migration (TEM). TEM involves several well-studied sequential adhesive interactions with vascular endothelial cells (ECs); however, what initiates or terminates this process is not well-understood. Here, we describe what we believe to be a new mechanism where vessel-associated macrophages through localized interactions primed EC responses to form ICAM-1 “hot spots” to support PMN TEM. Using real-time intravital microscopy of LPS-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we found that macrophage vessel association is critical for the initiation of PMN-EC adhesive interactions, PMN TEM, and subsequent accumulation in the intestinal mucosa. Anti–colony stimulating factor 1 receptor Ab-mediated macrophage depletion in the lamina propria and at the vessel wall resulted in elimination of ICAM-1 hot spots impeding PMN-EC interactions and TEM. Mechanistically, the use of human clinical specimens, TNF-α–KO macrophage chimeras, TNF-α/TNF receptor (TNF-α/TNFR) neutralization, and multicellular macrophage-EC-PMN cocultures revealed that macrophage-derived TNF-α and EC TNFR2 axis mediated this regulatory mechanism and was required for PMN TEM. As such, our findings identified clinically relevant mechanisms by which macrophages regulate PMN trafficking in inflamed mucosa.

Abstract Mass drug administration (MDA) of antimicrobials has shown promise in the reduction and potential elimination of a variety of neglected tropical diseases (NTDs). However, with antimicrobial resistance (AMR) becoming a global crisis, the risks posed by widespread antimicrobial use need to be evaluated. As the role of the environment in AMR emergence and dissemination has become increasingly recognized, it is likewise crucial to establish the role of MDA in environmental AMR pollution, along with the potential impacts of such pollution. This review presents the current state of knowledge on the antimicrobial compounds, resistant organisms, and antimicrobial resistance genes in MDA trials, routes of these determinants into the environment, and their persistence and ecological impacts, particularly in low and middle-income countries where these trials are most common. From the few studies directly evaluating AMR outcomes in azithromycin MDA trials, it is becoming apparent that MDA efforts can increase carriage and excretion of resistant pathogens in a lasting way. However, research on these outcomes for other antimicrobials used in MDA trials is sorely needed. Furthermore, while paths of AMR determinants from human waste to the environment and their persistence thereafter are supported by the literature, quantitative information on the scope and likelihood of this is largely absent. We recommend some mitigative approaches that would be valuable to consider in future MDA efforts. This review stands to be a valuable resource for researchers and policymakers seeking to evaluate the impacts of MDA. Graphical Abstract

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental–cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy. Video Abstract:

In species where offspring survival is highly variable relative to adult survival, such as bighorn sheep (Ovis canadensis), physiological indicators of maternal investment could clarify the functional mechanisms of life history trade-offs and serve as important predictors of population dynamics. From a management perspective, simple predictors of juvenile survival measured non-lethally from maternal samples could aid in identifying at-risk populations or individuals before significant mortality occurs. Blood biochemical parameters can offer low-cost insights into animal health and physiology, therefore we sought to develop a simple biochemical predictor of juvenile survival based on maternal blood samples. We measured biochemical indicators of energy balance in adult bighorn sheep at a single time point in January or February, and then monitored survival through August of the same year to assess how those measures related to survival of individual adults and their juvenile offspring. Juvenile survival was lower over the subsequent spring and summer when maternal adult serum beta-hydroxybutyric acid (β-HBA) concentration was high, indicating a negative energy balance in the mothers. However, serum β-HBA did not correlate with adult survival over the same period. Our findings suggest that even when maternal body condition is high, short-term caloric deficit may be sufficient trigger to decrease investment in offspring survival. This mechanism could protect adult females from investing heavily in juvenile survival when resources become too limited to support population growth. Our study suggests that β-HBA could be a powerful monitoring tool for bighorn sheep and other threatened ruminant populations under resource limitation.

Objective: Serotonin (5HT) is a well-known anorexigenic molecule, and 5HT neurons of dorsal raphe nucleus (DRN) have been implicated in suppression of feeding; however, the downstream circuitry is poorly understood. Here we explored major projections of DRN5HT neurons for their capacity to modulate feeding. Methods: We used optogenetics to selectively activate DRN5HT axonal projections in hypothalamic and extrahypothalamic areas and monitored food intake. We next used fiber photometry to image the activity dynamics of DRN5HT axons and 5HT levels in projection areas in response feeding and metabolic hormones. Finally, we used electrophysiology to determine how DRN5HT axons affect downstream neuron activity. Results: We found that selective activation of DRN5HT axons in (DRN5HT → LH) and (DRN5HT → BNST) suppresses feeding whereas activating medial hypothalamic projections has no effect. Using in vivo imaging, we found that food access and satiety hormones activate DRN5HT projections to LH where they also rapidly increase extracellular 5HT levels. Optogenetic mapping revealed that DRN5HT → LHvGAT and DRN5HT → LHvGlut2 connections are primarily inhibitory and excitatory respectively. Further, in addition to its direct action on LH neurons, we found that 5HT suppresses GABA release from presynaptic terminals arriving from AgRP neurons. Conclusions: These findings define functionally redundant forebrain circuits through which DRN5HT neurons suppress feeding and reveal that these projections can be modulated by metabolic hormones.

Background: The teres minor (TMi) muscle exposed relatively high activity during the acceleration and deceleration phases of the throwing motion, compared with the infraspinatus muscle. However, few studies have identified TMi muscle activity in intervention exercises. The purpose of this study was to investigate TMi muscle activities in different horizontal adduction positions in the quadruped horizontal abduction exercise. This study hypothesized that TMi muscle activity would differ in response to resistance application across different horizontal adduction positions. Materials and methods: Nineteen collegiate baseball players volunteered their participation. Raw electromyography activity of the TMi muscle along with 7 different muscles attached to the scapula on the dominant-side were collected, and normalized by each of the corresponding maximum voluntary isometric contractions. All subjects performed manual isometric resistance horizontal abduction exercises at 90° and 135° of abduction with 3 horizontal adduction angles in the quadruped position: 1) coronal, 2) scapular, and 3) sagittal plane. Electromyography data were also collected from rhythmical concentric contraction of horizontal abduction at 90° of abduction in the quadruped position. Results: TMi muscle activity was significantly greater with the arm positioned in the coronal plane than that of the scapular and sagittal planes (41, 26, and 17% maximum voluntary isometric contraction, respectively) (P

Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.

Blue light (400–500 nm) alleviates overexposure risks associated to UV light and has therefore gained increased interest in multiple applications. This meta-analysis deals with decontamination of E. coli through the use of blue light based from nine recent publications identified via a systematic literature search. In these studies, various pathogenic and non-pathogenic E. coli strains grown in nutritional broths were exposed to wavelengths ranging from 395 to 460 nm. Five meta-analyses were performed using Cochrane’s software for meta-analyses (Review Manager): one including all studies to estimate the effect of E. coli reduction and four subgroup-analyses considering reported intensities, wavelengths, exposure dose as well as serovars/pathovars. Random effects models were used. All included studies used colony-forming units to estimate the impact of E. coli reduction. None of the included studies involved an organic matrix (e.g., skin, food related surface). Exposure to blue light had a significant and large reducing effect on viable counts of E. coli. However, substantial heterogeneity across studies was observed. Among subgroups, reported intensity and wavelength showed the clearest impact on E. coli reduction. With respect to the reported exposure dose, the picture across the spectrum was scattered, but effect sizes tend to increase with increasing exposure dose. Substantial heterogeneity was also present with respect to all serovar/pathovar subgroups among the included studies. The present body of reports does not display a strong basis for recommendation of relevant intensities, wavelengths and exposure doses for superficial blue light decontamination in medical or food safety contexts. A serious shortcoming in most studies is the absence of a clear documentation of inoculum preparation and of study parameters. We suggest improvement for study protocols for future investigations.

Cardiovascular disease remains a leading cause of morbidity and mortality worldwide. Aspects of disease severity that are associated with heightened inflammation, such as during atherosclerosis or after myocardial infarction, are correlated with macrophage activation and macrophage polarization of the transcriptome and secretome. In this setting, non-coding RNAs (ncRNAs) may be as abundant as protein-coding genes and are increasingly recognized as significant modulators of macrophage gene expression and cytokine secretion, although the functions of most ncRNAs—and in particular, long non-coding RNAs—remain unknown. Herein, we discuss a subset of specific ncRNAs of interest in macrophages in atherosclerosis and during myocardial inflammation.

Abstract Background Helicobacter pylori is a Gram-negative bacterium which mainly causes peptic ulcer disease in human, but is also the predominant cause of stomach cancer. It has been coevolving with human since 120,000 years and, according to Multi-locus sequence typing (MLST), H. pylori can be classified into seven major population types, namely, hpAfrica1, hpAfrica2, hpNEAfrica, hpEastAsia, hpAsia2, hpEurope and hpSahul. Helicobacter pylori harbours a large number of restriction-modification (R-M) systems. The methyltransferase (MTase) unit plays a significant role in gene regulation and also possibly modulates pathogenicity. The diversity in MTase can act as geomarkers to correlate strains with the phylogeographic origins. This paper describes the complete genome sequence and methylome of gastric pathogen H. pylori belonging to the population hpNEAfrica. Results In this paper, we present the complete genome sequence and the methylome profile of H. pylori hpNEAfrica strain HP14039, isolated from a patient who was born in Somalia and likely to be infected locally during early childhood prior to migration. The genome of HP14039 consists of 1,678,260 bp with 1574 coding genes and 38.7% GC content. The sequence analysis showed that this strain lacks the cag pathogenicity island. The vacA gene is of S2M2 type. We have also identified 15 methylation motifs, including WCANHNNNNTG and CTANNNNNNNTAYG that were not previously described. Conclusions We have described the complete genome of H. pylori strain HP14039. The information regarding phylo-geography, methylome and associated metadata would help scientific community to study more about hpNEAfrica population type.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

Cardiovascular disease remains the leading cause of death worldwide. Myocardial ischemia is a major contributor to cardiovascular morbidity and mortality. In the case of acute myocardial infarction, subsequent cardiac repair relies upon the acute, and coordinated response to injury by innate myeloid phagocytes. This includes neutrophils, monocytes, macrophage subsets, and immature dendritic cells. Phagocytes function to remove necrotic cardiomyocytes, apoptotic inflammatory cells, and to remodel extracellular matrix. These innate immune cells also secrete cytokines and growth factors that promote tissue replacement through fibrosis and angiogenesis. Within the injured myocardium, macrophages polarize from pro-inflammatory to inflammation-resolving phenotypes. At the core of this functional plasticity is cellular metabolism, which has gained an appreciation for its integration with phagocyte function and remodeling of the transcriptional and epigenetic landscape. Immunometabolic rewiring is particularly relevant after ischemia and clinical reperfusion given the rapidly changing oxygen and metabolic milieu. Hypoxia reduces mitochondrial oxidative phosphorylation and leads to increased reliance on glycolysis, which can support biosynthesis of pro-inflammatory cytokines. Reoxygenation is permissive for shifts back to mitochondrial metabolism and fatty acid oxidation and this is ultimately linked to pro-reparative macrophage polarization. Improved understanding of mechanisms that regulate metabolic adaptations holds the potential to identify new metabolite targets and strategies to reduce cardiac damage through nutrient signaling.

We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD “ene” reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.

Objective: Genomewide association studies (GWAS) have identified consistent associations with obesity, with a number of studies implicating eating behavior as a primary mechanism. Few studies have replicated genetic associations with dietary intake. This study evaluates the association between obesity susceptibility loci and dietary intake., Methods: Data were obtained as part of the Diabetes Prevention Program (DPP), a clinical trial of diabetes prevention in persons at high risk of diabetes. The association of 31 genomewide association studies identified obesity risk alleles with dietary intake, measured through a food frequency questionnaire, was investigated in 3,180 participants from DPP at baseline., Results: The minor allele at BDNF, identified as protective against obesity, was associated with lower total caloric intake (β = -106.06, SE = 33.13; p = .0014) at experimentwide statistical significance (p = .0016), whereas association of MC4R rs571312 with higher caloric intake reached nominal significance (β = 61.32, SE = 26.24; p = .0194). Among non-Hispanic white participants, the association of BDNF rs2030323 with total caloric intake was stronger (β = -151.99, SE = 30.09; p < .0001), and association of FTO rs1421085 with higher caloric intake (β = 56.72, SE = 20.69; p = .0061) and percentage fat intake (β = 0.37, SE = 0.08; p = .0418) was also observed., Conclusions: These results demonstrate with the strength of independent replication that BDNF rs2030323 is associated with 100 to 150 greater total caloric intake per allele, with additional contributions of MC4R and, in non-Hispanic white individuals, FTO. As it has been argued that an additional 100 kcal/d could account for the trends in weight gain, prevention focusing on genetic profiles with high dietary intake may help to quell adverse obesity trends., Clinical Trial Registration: Clinicaltrials.gov,NCT00004992., Competing Interests: LMD has a financial interest in Omada Health, a company that develops online behavior-change programs, with a focus on diabetes. LMD's interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. No other potential conflict of interest is declared by any author.

Current FDA regulatory screening and confirmatory methods, electronic cell counting and the direct microscopic somatic cell count (DMSCC), differ for the detection of abnormal milk in sheep and goats. The DNA-specific electronic SCC screening methods such as Fossomatic (Foss, Hillerød, Denmark) can be used for both sheep and goat milk; however, the nonspecific methylene blue-based stains used for DMSCC in sheep cannot be used for goats as they nonspecifically stain cytoplasmic particles naturally present in goat milk. The DNA-specific stain pyronin Y-methyl green (PMG) is currently used for DMSCC in goats. Sheep also shed cytoplasmic particles during the milk secretory process, but in fewer numbers than goats. The objective of this study was to determine whether the nonspecific, methylene blue-based Levowitz-Weber (L-W) stain is the appropriate regulatory stain to use for DMSCC in sheep milk. Composite milk samples from 42 commercial dairy ewes were collected every 4 wk for the duration of each ewe's lactation for a total of 10 sample days. Milk samples were subjected to 3 methods of SCC determination: automated Fossomatic counting, DMSCC with L-W stain, and DMSCC with PMG stain conducted according to FDA regulatory procedures (2400 series forms). The DMSCC from milk smears stained with L-W were greater than those from smears stained with PMG and those from the Fossomatic analysis on 6 of the 10 sampling days. Milk smears stained with PMG did not differ from Fossomatic analysis at any sampling. The average milk SCC for L-W, PMG, and Fossomatic were (mean±SE) 275±36×10(3), 174±24×10(3), and 164±24×10(3) cells/mL, respectively. The DMSCC for milk stained with L-W was 58% greater than that with PMG and 68% greater than that obtained with the Fossomatic analysis. In conclusion, DMSCC of sheep milk stained with the nonspecific, methylene blue L-W stain resulted in a marked increase in SCC over that of the DNA-specific stain PMG and Fossomatic SCC analysis. The findings of this study support the continued use of Fossomatic SCC but recommend the replacement of the methylene blue-based stains with DNA-specific PMG for determination of DMSCC in sheep milk., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)