CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells.

The objective of this study was to determine the role of GCN2 in the response to AA deprivation of primary bovine mammary epithelial cells (BMEC). Cells were isolated from the mammary tissue of 2 lactating Holstein cows by enzymatic digestion, expanded, and induced to differentiate for 5 to 7 d. Relative mRNA expression was measured by real-time quantitative PCR. Protein abundance and site-specific phosphorylation were measured by immunoblotting. Knockout of GCN2 in BMEC was accomplished by lentiviral delivery of a targeted single guide RNA and endonuclease Cas9. To investigate the role of GCN2, we treated lactogenic differentiated BMEC with either culture medium lacking Arg, Leu, and Lys combined or lacking only one of the 3 AA of interest, in comparison to a control with a full complement of AA. Activation of GCN2 was inferred by the phosphorylation status of its downstream target eIF2α Ser51. We found that GCN2 was activated by both the deprivation of Arg, Leu, and Lys combined and of Arg alone, as shown by a 2.73- and 2.82-fold increase in phosphorylated eIF2α Ser51 after 1 h of deprivation, respectively. In addition, activation of GCN2 as measured by increased phosphorylation of eIF2α Ser51 during the deprivation of Arg, Leu, and Lys combined and of Arg alone was sustained for up to 8 h of deprivation. Phosphorylated eIF2α selectively upregulates translation of transcription factor ATF4, among others, during AA deprivation, which then targets genes necessary for restoring AA homeostasis. Therefore, we investigated the expression of ATF4 transcriptional targets, AA enzyme ASNS and AA transporters SLC7A1 and SLC38A2. We found that ASNS was upregulated in response to combined AA deprivation and by Arg deprivation alone by 3.6- and 4.51-fold, respectively, at 24 h of treatment. We found that SLC7A1 was upregulated in response to combined AA deprivation and deprivation of Arg alone by 2.0- and 2.36-fold, respectively, at 8 h of treatment. To establish the role of GCN2 (encoded by EIF2AK4) in the response to AA deprivation, we ablated GCN2 in BMEC using clustered regularly interspaced short palindromic repeats-Cas9. We showed that BMEC transduced with single guide RNA targeting EIF2AK4 were not as responsive to combined AA deprivation, compared with BMEC transduced with nontargeting single guide RNA. Taken together, our results demonstrate a critical role for GCN2 in sensing AA deprivation in BMEC.

Martial arts as sport and therapy.

The term Martial Arts is often used as general phrase to describe many of the combat arts, which have developed in eastern cultures over the past millennium. This paper reviews the Martial Arts from the original context of a trio of life skills. This trio includes the healing arts such as acupuncture, the self-exploration arts such as yoga, and the vital life skills such as meditation. As Martial Arts suggests the waging of combat, the origins of the most common combat arts are reviewed, with an overview of the difference between the hard and the soft styles. The arts developed not only in the eastern, but also in all parts of the world, with references of these types of combats arts in the writings of the ancient Egyptians and Greeks. In modern times, the combat arts are performed for both exercise and sport. A review of the injuries that occur, and the health benefits that might be expected are discussed. A review of the medical literature that demonstrates some of these health benefits is included, with Tai Chi Chuan as the most studied of these. The health benefits discussed include strengthen and self-efficacy of the elderly, reduced falls, increased exercise capacity, and benefits to the immune system and autonomic nervous system. The paper emphasized the breadth of the Martial Arts and the import of these to the sports and health community.

Hypothalamus and amygdala response to acupuncture stimuli in Carpal Tunnel Syndrome.

Brain processing of acupuncture stimuli in chronic neuropathic pain patients may underlie its beneficial effects. We used fMRI to evaluate verum and sham acupuncture stimulation at acupoint LI-4 in Carpal Tunnel Syndrome (CTS) patients and healthy controls (HC). CTS patients were retested after 5 weeks of acupuncture therapy. Thus, we investigated both the short-term brain response to acupuncture stimulation, as well as the influence of longer-term acupuncture therapy effects on this short-term response. CTS patients responded to verum acupuncture with greater activation in the hypothalamus and deactivation in the amygdala as compared to HC, controlling for the non-specific effects of sham acupuncture. A similar difference was found between CTS patients at baseline and after acupuncture therapy. For baseline CTS patients responding to verum acupuncture, functional connectivity was found between the hypothalamus and amygdala--the less deactivation in the amygdala, the greater the activation in the hypothalamus, and vice versa. Furthermore, hypothalamic response correlated positively with the degree of maladaptive cortical plasticity in CTS patients (inter-digit separation distance). This is the first evidence suggesting that chronic pain patients respond to acupuncture differently than HC, through a coordinated limbic network including the hypothalamus and amygdala.

Injection of the piriformis muscle by fluoroscopic and electromyographic guidance.

BACKGROUND: There is not a universally accepted single technique for injection of the piriformis muscle that has validated exact placement of the needle tip within the piriformis muscle. OBJECTIVE: We sought a methodology that would precisely document needle placement within the piriformis muscle that is reliable, relatively uncomplicated, and reproducible. METHODS: Patients with piriformis syndrome underwent injections of the piriformis muscle under fluoroscopic and electromyographic guidance. This technique used electrophysiological confirmation of needle placement within the piriformis muscle and image-guided identification of the piriformis muscle with radiopaque contrast media under fluoroscopy. RESULTS: Using this methodology, injections on 17 occasions in 11 patients resulted in needle placement within the piriformis muscle.

Ultrastructural and functional analyses of nephropathy in calmodulin-induced diabetic transgenic mice.

BACKGROUND: Previous animal models of diabetic nephropathy have used diabetic animals for which the underlying defect was either uncertain or the diabetes was induced by potentially specific toxins. In this report, we describe the renal abnormalities in a transgenic mouse model that develops early-onset diabetes due to overexpression of calmodulin in pancreatic beta cells. METHODS: Renal tissues were collected from normal and transgenic mice at 112, 182, and 300 days. These were prepared for light microscopic observation, stained with polyethylenimine (for anionic sites), or rendered acellular by detergent extraction prior to observation by transmission and scanning electron microscopy. Morphometric analysis of glomerular basement membrane thickness was carried out by the "orthogonal intercept" method. Twelve-hour urine samples of fed and fasting mice were collected for urine volume and glucose and protein analyses. Blood glucose, blood urea nitrogen, serum insulin, and creatinine were determined in 60-90-day-old and 255-day-old mice by established methods. RESULTS: Morphometric analyses revealed age-related and transgene-related increases in glomerular basement membrane thickness. A 22% increase in transgenic diabetics over controls was seen at 112 days of age that developed to increases of 43% and 37% at 182 and 300 days of age, respectively. Mesangial matrix area was also increased markedly in transgenic mice. Surprisingly, even in the oldest diabetic mice, there was no reduction in anionic sites. Moreover, despite an eightfold increase in urine volume, these mice did not become significantly proteinuric. CONCLUSIONS: These results indicate that proteinuria of diabetes may be delayed or prevented by maintenance of a normal complement of glomerular basement membrane anionic sites. They also demonstrate that transgenic mice can provide a valuable model for discriminating between different aspects of diabetic nephropathy.

Characterization of immunomodulatory properties and accessory cell function of small intestinal epithelial cells.

We have studied the immunomodulatory properties of epithelial cells from the small intestine on T cell immune function in vitro. Proliferation of lymph node cells stimulated either with antigen or with mitogen was inhibited by epithelial cells in a dose-dependent fashion. The epithelial cell-mediated suppression of lymphocyte proliferation was blocked by indomethacin, a cyclooxygenase pathway inhibitor, demonstrating that the suppressive effect of epithelial cells was related to prostaglandin secretion. Furthermore, the action of epithelial cell-secreted prostaglandin on lymphocytes was related to its effect on IL-2 as the suppressive effect of epithelial cells was abrogated by the addition of exogenous IL-2. As previously reported, epithelial cells constitutively express MHC class II and we found them able to present antigen in a class II-restricted fashion when their suppressive effects were blocked by indomethacin. Furthermore, epithelial cells activated by LPS secrete an IL-1 like molecule in a fashion analogous to other antigen-presenting cells. These results demonstrate that epithelial cells can both enhance and suppress in vitro T cell immune responses and further characterize the mechanisms by which intestinal epithelial cells may function in gut-associated immune responses.

Intrinsic fibrillar components of human glomerular basement membranes: a TEM analysis following proteolytic dissection.

At the level of ultrastructure, basement membranes (BMs) are usually described as thin layers of extracellular matrix comprised of an interwoven mat of fine 3-4 nm fibrils embedded in a granular matrix. In order to improve the resolution of the fibrillar components, we have carried out TEM studies on human glomerular BMs (GBMs) made acellular by sequential detergent solubilization. Some GBMs were pretreated with pronase, trypsin, or pepsin for 30 min to 72 h prior to preparation for microscopy. Our study shows that irrespective of which enzyme is employed, background granular matrix is first solubilized leaving a three dimensional fibrillar network comprised of 3-8 nm fibrils. Larger 7-8 nm fibrils are concentrated near subepithelial portions of the GBM and are most resistant to proteolysis. Smaller 3-4 nm fibrils are located primarily subjacent to endothelium and mesangial cells and are more protease-sensitive. An unexpected finding in pepsinized samples was a quasi-hexagonal fibrillocrystalline structure associated with mesangial matrix and subendothelial portions of the GBM. These data suggest that intrinsic fibrillar components of human GBMs are heterogeneously distributed throughout the thickness of their laminae densae. We speculate that the network consists of type IV collagen and that the hexagonal crystals may represent type VI or some previously unreported BM collagen type.

Ultrastructural evidence for morphological specificity in isolated bovine retinal capillary basement membranes.

Because of its relative availability, large size, and presumed similarity to human, the bovine retina has been used by numerous investigators as a source of vessels, cells, and basement membranes (BMs) for biochemical analyses and in vitro studies of cells and extracellular matrix. Careful morphological studies of these vessels and their associated BMs, however, have not been done. Accordingly, we carried out experimental ultrastructural studies in an effort to show their cellular composition, their histoarchitectural relationships within retinal capillary walls, and the disposition and features of their isolated BMs. Our study shows that these vessels are complex, multicomponent structures composed of endothelial cells and intramural pericytes, which frequently communicate via direct cell/cell contacts, and a system of BMs. The latter includes continuous Muller cell BMs, interrupted subendothelial BMs, and pericytic BMs with masses of pericytic matrix (PCM) intervening. Isolated subendothelial BMs are remarkable for fenestrations, selective susceptibility to nonspecific proteases, and high density of ruthenium red (RR)-positive anionic sites. On the contrary, Muller cell BMs are continuous (completely surrounding retinal capillaries), relatively refractory to proteases, and show significantly fewer anionic sites by RR. Acellular capillary BMs frequently show ghost-like "pockets" previously occupied by pericytes. These are surrounded by pericytic BMs and interstitial spaces are "filled-in" by a BM-like material (PCM) which frequently contains striated collagen fibrils and is positionally and morphologically homologous to glomerular mesangial matrix. These data indicate that tissue specificity of BMs may be far more precise than previously thought and that each capillary BM leaflet may possess a peculiar macromolecular architecture commensurate with its specific function.