Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.

Leptin and its long form receptor, Ob-Rb, in the hypothalamus play a key function in regulating food intake and body weight. We demonstrate here that apolipoprotein D (Apo D), a lipocalin that is heavily expressed in brain, interacts specifically with the cytoplasmic portion of Ob-Rb but not the short form receptor, Ob-Ra. This finding is detected in yeast two-hybrid systems as well as in protein precipitation experiments in vitro and in vivo. Apo D and Ob-Rb are coexpressed in neurons of the hypothalamic arcuate and paraventricular nuclei that are known to be involved in eating and body weight regulation. Moreover, hypothalamic level of Apo D mRNA is stimulated by dietary fat. It is also significantly elevated in rats and inbred mice that become obese on a highfat diet, compared with their lean controls, and is strongly, positively correlated with body fat mass and circulating leptin levels. This positive association with body fat, however, is lost in obese ob-/ob-and db-/db-mice, which exhibit markedly reduced levels of hypothalamic Apo D mRNA compared with that of wild-type mice. These results suggest that Apo D in the hypothalamus is involved in the leptin/Ob-Rb signal transduction pathway that controls body fat accumulation on a high-fat diet.Key words: apolipoprotein D • leptin • leptin receptor • obesity • diabetes • signal transduction. polipoprotein D (Apo D) was originally purified from blood as a minor apoprotein component of the high-density lipoprotein particles and was thus designated as apolipoprotein (1). However, Apo D does not show sequence similarity to other apolipoproteins (2). Instead, it is a member of the lipocalin family of proteins that are characterized by a conserved eight-stranded, beta-barrel tertiary structure and function in the binding and transporting of small hydrophobic ligands (3). The physiological function of Apo D, as well as the identity of its putative ligand, remains to be characterized. It has been proposed that Apo D is a transporter for an unknown ligand that is structurally similar to heme-related molecules, such as biliverdin and porphyrins (4). In addition, Apo D might bind and transport other ligands, including arachidonic acid, cholesterol, and steroid hormones such as progesterone and pregnenolone (2,5). This ability of Apo D to associate with a variety of ligands suggests that it might have multiple, tissue-specific functions. For example, Apo D binds progesterone in breast A cyst fluid (6), and it also binds and transports an axilliary odorant when it is secreted from apocrine glands (7).Although it is present in a wide range of peripheral tissues in primates and rabbits (8), Apo D is highly concentrated in brain and is almost undetectable in most peripheral tissues in rodents (9). It is reported that, in brain, Apo D is expressed in the parenchyma, as well as in the subarachnoid space and pia matter, and it is synthesized in a number of cell types, including neurons, neuroglial cells, astrocytes, fibroblasts, and perivascular cells (10, 11). There is evidence...

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Leptin and its long form receptor, Ob-Rb, in hypothalamic nuclei play a key role in regulating energy balance. The mutation of Ob-Rb into one of its natural variants, Ob-Ra, results in severe obesity in rodents. We demonstrate here that diacylglycerol kinase (DGK) interacts, via its ankyrin repeats, with the cytoplasmic portion of Ob-Rb in yeast two-hybrid systems, in protein precipitation experiments in vitro and in vivo. It does not interact, however, with the short form, Ob-Ra, which mediates the entry of leptin into the brain. Furthermore, we show by in situ hybridization that DGK is expressed in neurons of hypothalamic nuclei known to synthesize Ob-Rb and to participate in energy homeostasis. The mutant ob-/ob-and db-/db-mice exhibit increased hypothalamic DGK mRNA level compared with their wild-type controls, suggesting a role for the leptin/ OB-Rb system in regulating DGK expression. Further experiments show that hypothalamic DGK mRNA level is stimulated by the consumption of a high-fat diet. In addition, DGK mRNA is statistically significantly lower in rats and inbred mice that become obese on a high-fat diet compared with their lean counterparts. In fact, it is strongly, negatively correlated with both body fat and circulating levels of leptin. Taken together, our evidence suggests that DGK constitutes a downstream component of the leptin signaling pathway and that reduced hypothalamic DGK mRNA, and possibly activity, is associated with obesity.

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