Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

Adipose differentiation is accompanied by changes in cellular morphology, a dramatic accumulation of intracellular lipid and activation of a specific program of gene expression. Using an mRNA differential display technique, we have isolated a novel adipose cDNA, termed adipoQ. The adipoQ cDNA encodes a polypeptide of 247 amino acids with a secretory signal sequence at the amino terminus, a collagenous region (Gly-X-Y repeats), and a globular domain. The globular domain of adipoQ shares significant homology with subunits of complement factor C1q, collagen ␣1(X), and the brainspecific factor cerebellin. The expression of adipoQ is highly specific to adipose tissue in both mouse and rat. Expression of adipoQ is observed exclusively in mature fat cells as the stromal-vascular fraction of fat tissue does not contain adipoQ mRNA. In cultured 3T3-F442A and 3T3-L1 preadipocytes, hormone-induced differentiation dramatically increases the level of expression for adipoQ. Furthermore, the expression of adipoQ mRNA is significantly reduced in the adipose tissues from obese mice and humans. Whereas the biological function of this polypeptide is presently unknown, the tissue-specific expression of a putative secreted protein suggests that this factor may function as a novel signaling molecule for adipose tissue.Adipose tissue is highly specialized to play important roles in energy storage, fatty acid metabolism, and glucose homeostasis (1, 2). Adipocytes synthesize and store triglyceride in periods of nutritional abundance and mobilize the lipids in response to fasting (2, 3). Fat tissue is also involved in regulating blood glucose levels through the expression of the insulin responsive glucose transporter, Glu4 (4, 5). Fat and muscle, in fact, constitute the two major sites for insulin-regulated glucose uptake.At a molecular level, many genes involved in lipid metabolism and glucose homeostasis are prominently expressed in fat (1). These include fatty acid synthase (6), the fatty acid binding protein aP2 (7,8), lipoprotein lipase (9), phosphoenolpyruvate carboxykinase (10), malic enzyme (11), glyceraldehyde-3-phosphate dehydrogenase (12), and Glut4 (4). Receptors for lipogenic or lipolytic hormones such as insulin (13, 14), insulin-like growth factor 1 (15), and adrenergic compounds (16,17) are also expressed in adipocytes. In addition to these genes that clearly participate in the metabolic functions of adipose tissue, a group of genes that function in extracellular signaling have also been identified in fat. A prototype of these molecules is insulin-like growth factor 1, which is expressed in many tissues during development and plays an important role in cell proliferation (18). In adipocytes, however, insulin-like growth factor 1 is found to stimulate cell differentiation (19). More interestingly, insulin-like growth factor 1 is synthesized by preadipocytes in response to growth hormone stimulation (20), thus potentially functioning in an autocrine or paracrine fashion to promote adipogenesis during development. Another si...

Astrocytic energy demand is stimulated by K + and glutamate uptake, signaling processes, responses to neurotransmitters, Ca 2 + fluxes, and filopodial motility. Astrocytes derive energy from glycolytic and oxidative pathways, but respiration, with its high-energy yield, provides most adenosine 5 0 triphosphate (ATP). The proportion of cortical oxidative metabolism attributed to astrocytes (B30%) in in vivo nuclear magnetic resonance (NMR) spectroscopic and autoradiographic studies corresponds to their volume fraction, indicating similar oxidation rates in astrocytes and neurons. Astrocyte-selective expression of pyruvate carboxylase (PC) enables synthesis of glutamate from glucose, accounting for two-thirds of astrocytic glucose degradation via combined pyruvate carboxylation and dehydrogenation. Together, glutamate synthesis and oxidation, including neurotransmitter turnover, generate almost as much energy as direct glucose oxidation. Glycolysis and glycogenolysis are essential for astrocytic responses to increasing energy demand because astrocytic filopodial and lamellipodial extensions, which account for 80% of their surface area, are too narrow to accommodate mitochondria; these processes depend on glycolysis, glycogenolysis, and probably diffusion of ATP and phosphocreatine formed via mitochondrial metabolism to satisfy their energy demands. High glycogen turnover in astrocytic processes may stimulate glucose demand and lactate production because less ATP is generated when glucose is metabolized via glycogen, thereby contributing to the decreased oxygen to glucose utilization ratio during brain activation. Generated lactate can spread from activated astrocytes via low-affinity monocarboxylate transporters and gap junctions, but its subsequent fate is unknown. Astrocytic metabolic compartmentation arises from their complex ultrastructure; astrocytes have high oxidative rates plus dependence on glycolysis and glycogenolysis, and their energetics is underestimated if based solely on glutamate cycling. Keywords: acetate; glucose metabolism; glutamate; neurotransmitters; potassium; pyruvate carboxylation Introduction Astrocytes are More Than HousekeepersRecent studies in many different fields have shown much more active roles of astrocytes in brain function than previously portrayed by their traditionally ascribed 'bystander or housekeeping' functions. Emerging roles of astrocytes include their interactions with the vasculature, neurons, and other astrocytes via signaling, biosynthetic, and transport processes to regulate blood flow, modulate impulse transmission, and synthesize and degrade glucose-derived neurotransmitters, for example, glutamate and g-aminobutyric acid (GABA) (Takano et al, 2006;Hertz and Zielke, 2004;Volterra and Meldolesi, 2005). All of these processes are energyrequiring or dependent on energy-related metabolic pathways, thereby directly linking astrocyte functions, energetics, and metabolite fluxes.The narrow astrocytic surface extensions (lamellae and filopodia, also called peripheral ast...

Differential display has been developed as a tool to detect and characterize altered gene expression in eukaryotic cells. The basic principle is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Here we provide methodological details and examine in depth the specificity, sensitivity and reproducibility of the method. We show that the number of anchored oligo-dT primers can be reduced from twelve to four that are degenerate at the penultimate base from the 3' end. We also demonstrate that using optimized conditions described here, multiple RNA samples from related cells can be displayed simultaneously. Therefore process-specific rather than cell-specific genes could be more accurately identified. These results enable further streamlining of the technique and make it readily applicable to a broad spectrum of biological systems.

Although airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been recognized, the condition of ventilation for its occurrence is still being debated. We analyzed a coronavirus disease 2019 (COVID-19) outbreak involving three families in a restaurant in Guangzhou, China, assessed the possibility of airborne transmission, and characterized the associated environmental conditions. We collected epidemiological data, obtained a full video recording and seating records from the restaurant, and measured the dispersion of a warm tracer gas as a surrogate for exhaled droplets from the index case. Computer simulations were performed to simulate the spread of fine exhaled droplets. We compared the in-room location of subsequently infected cases and spread of the simulated virus-laden aerosol tracer. The ventilation rate was measured using the tracer gas concentration decay method. This outbreak involved ten infected persons in three families (A, B, C). All ten persons ate lunch at three neighboring tables at the same restaurant on January 24, 2020. None of the restaurant staff or the 68 patrons at the other 15 tables became infected. During this occasion, the measured ventilation rate was 0.9 L/s per person. No close contact or fomite contact was identified, aside from back-to-back sitting in some cases. Analysis of the airflow dynamics indicates that the infection distribution is consistent with a spread pattern representative of long-range transmission of exhaled virus-laden aerosols. Airborne transmission of the SARS-CoV-2 virus is possible in crowded space with a ventilation rate of 1 L/s per person.

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words)Main text (3456 words) AbstractBackground: The role of aerosols in the transmission of SARS-CoV-2 remains debated. We analysed an outbreak involving three non-associated families in Restaurant X in Guangzhou, China, and assessed the possibility of aerosol transmission of SARS-CoV-2 and characterize the associated environmental conditions. : medRxiv preprint 2 Methods: We collected epidemiological data, obtained a video record and a patron seatingarrangement from the restaurant, and measured the dispersion of a warm tracer gas as a surrogate for exhaled droplets from the suspected index patient. Computer simulations were performed to simulate the spread of fine exhaled droplets. We compared the in-room location of subsequently infected cases and spread of the simulated virus-laden aerosol tracer. The ventilation rate was measured using the tracer decay method.Results: Three families (A, B, C), 10 members of which were subsequently found to have been infected with SARS-CoV-2 at this time, or previously, ate lunch at Restaurant X on Chinese New Year's Eve (January 24, 2020) at three neighboring tables. Subsequently, three members of family B and two members of family C became infected with SARS-CoV-2, whereas none of the waiters or 68 patrons at the remaining 15 tables became infected. During this occasion, the ventilation rate was 0.75-1.04 L/s per person. No close contact or fomite contact was observed, aside from back-to-back sitting by some patrons. Our results show that the infection distribution is consistent with a spread pattern representative of exhaled virus-laden aerosols.Conclusions: Aerosol transmission of SARS-CoV-2 due to poor ventilation may explain the community spread of COVID-19.