Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P 2 , produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A smallinterfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P 2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P 2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P 2 synthesis and endomembrane homeostasis in mammalian cells.

The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.

Schools have been closed in many countries due to the on-going COVID-19 pandemic, but education continues online. Little is known about how parents cope with educating their children in this unprecedented situation. Here, we present the results of a rapid survey examining the experience of Czech parents of children in Grades 1-9 (Age ~ 6-15; N = 9,810) with respect to education at home during the COVID-19 lockdown. This survey was distributed widely, but only online and parents participated voluntarily. Mainly families with an internet connection and interested in their children’s education (i.e., the majority of families with school-aged children in the Czech Republic) took part in the survey. The results show that these families tend to cope well with the current educational situation and view the overall schoolwork transferred to homes as useful. Most children spend 2-4 hours a day studying, while parents help them at least half the time. Parents mostly explain task instructions, check the work their children have done, and teach new topics. To a lesser extent, they help their children solve tasks. Teachers appear to assign tasks more often than they provide feedback and/or interact with children. Some parents face difficulties, but those are generally not severe. These include, most notably, a lack of time, issues with technologies, and inadequate teaching skills and content knowledge. Altogether, this work maps the current educational situation in a large segment of Czech families and highlights possible pitfalls to be avoided: in the Czech Republic and beyond.

Schools have been closed in many countries due to the on-going COVID-19 pandemic, but education continues online. Little is known about how parents cope with educating their children in this unprecedented situation. Here, we present the results of a rapid survey examining the experience of Czech parents of children in Grades 1-9 (Age ∼ 6-15; N = 9,810) with respect to home education during the COVID-19 lockdown. This survey was distributed widely, but only online and parents participated voluntarily. Mainly families with an internet connection and interested in their children's education (i.e., the majority of families with school-aged children in the Czech Republic) took part in the survey. The results show that these families tend to cope well with the current educational situation and view the overall schoolwork transferred to homes as useful. Most children spend 2-4 h a day studying, while parents help them at least half the time. Parents mostly explain task instructions, check the work their children have done, and teach new topics. To a lesser extent, they help their children solve tasks. Teachers appear to assign tasks more often than they provide feedback and/or interact with children. Some parents face difficulties, but those are generally not severe. These include, most notably, a lack of time, issues with technologies, and inadequate teaching skills and content knowledge. Altogether, this work maps the current educational situation in a large segment of Czech families and highlights possible pitfalls to be avoided: in the Czech Republic and beyond.

Inhibitors and methyl donor substrates for betaine-homocysteine S-methyltransferase (BHMT) were used to study the role of this enzyme in the regulation of plasma total homocysteine (tHcy). Mice were administered an i.p. injection of S-(delta-carboxybutyl)-dl-homocysteine (CBHcy; 1 mg), a specific and potent inhibitor of BHMT, and tHcy and hepatic BHMT protein and activity levels were monitored over a 24-h period. Compared with saline-injected control mice, at 2 h postinjection, the CBHcy-treated mice had 87% lower BHMT activity and a 2.7-fold increase (11.1 vs. 3.0 micromol/L) in tHcy, effects that lasted nearly 8 h but returned to normal by 24 h. The level of BHMT protein remained constant over the 24-h period. After 6 CBHcy (1 mg) injections (one every 12 h), the mice had 7-fold higher tHcy, a 65% reduction in the liver S-adenosylmethionine:S-adenosylhomocysteine ratio, and a marked upregulation of BHMT protein expression. At 2 h after injection of the sulfoxide derivative of CBHcy (10 mg) into mice, there was a modest reduction in BHMT activity and a 90% increase in tHcy. When given an injection of Met (3 mg) or Met plus CBHcy (1 mg), post-Met load tHcy levels were 2.2-fold higher (128 vs. 40 micromol/L) at 2 h postinjection in the mice given CBHcy. Like betaine, dimethylsulfoniopropionate was an effective tHcy-lowering agent when given with a Met load. These studies are the first to show that transient inhibition of BHMT in vivo causes transient hyperhomocysteinemia, and that dimethylsulfoniopropionate can reduce a post-Met load rise in tHcy.