Scheduling Automatic Pickup by Self-driving Cars

This disclosure includes techniques to automatically schedule pickups by self-driving cars. The techniques utilize data about a user such as the user’s location, payments, calendar, to-do list etc. An example technique uses data from commercial establishments (e.g., payments systems), and self-driving cars to determine a location and a time at which the user needs to be picked up. A self-driving car system schedules a self-driving car to pick up the user at the specific location and time. The techniques described may be implemented in selfdriving cars, a central server system, user mobile devices, client devices, or different combinations of these systems and devices

FOLK MEDICINE AMONG TRIBE PEOPLE- A REVIEW

Today ancient Ayurvedic texts have been researched and a lot of hidden knowledge has been brought to surface light and Ayurveda is becoming one of the leading scientific way of preserving and promoting a healthy life. On the other-hand we do not know much about another type of medicine which is more ancient, equally efficacious and widely practised by its adherents: Folk medicines. Folk medicine was practised during Atharva-Vedic period and it is still practised in 21st century by tribal people, villagers and even by some people of cities who still adheres to their ancestor knowledge in India. Folk medicine has its own concept of causation of disease, diagnostic techniques and treatment. In history of Indian medicine little or almost no attention has been given to folk medicine, on the other-hand tribal and village people believe in folk medicine as it fits in with their culture and way of thinking. If we want that these people can derive benefit from Ayurveda or Modern medicine then we have to take pain to see that today’s medicine is presented to them in such a way that it is ‘acceptable’ to them. We can make it ‘acceptable’ only if we know what they believe in. So here effort was made to recollect knowledge about folk medicine in india so that it not remain patchy and inadequate and helps to promote healthy life

Leishmania highjack host lipid body for its proliferation in macrophages by overexpressing host Rab18 and TRAPPC9 by downregulating miR-1914-3p expression.

Lipids stored in lipid-bodies (LBs) in host cells are potential sources of fatty acids for pathogens. However, the mechanism of recruitment of LBs from the host cells by pathogens to acquire fatty acids is not known. Here, we have found that Leishmania specifically upregulates the expression of host Rab18 and its GEF, TRAPPC9 by downregulating the expression of miR-1914-3p by reducing the level of Dicer in macrophages via their metalloprotease gp63. Our results also show that miR-1914-3p negatively regulates the expression of Rab18 and its GEF in cells. Subsequently, Leishmania containing parasitophorous vacuoles (Ld-PVs) recruit and retain host Rab18 and TRAPPC9. Leishmania infection also induces LB biogenesis in host cells and recruits LBs on Ld-PVs and acquires FLC12-labeled fatty acids from LBs. Moreover, overexpression of miR-1914-3p in macrophages significantly inhibits the recruitment of LBs and thereby suppresses the multiplication of parasites in macrophages as parasites are unable to acquire fatty acids. These results demonstrate a novel mechanism how Leishmania acquire fatty acids from LBs for their growth in macrophages

Expression and recruitment of host Rab18 in <i>L</i>. <i>donovani</i> infected macrophages.

a, To determine the levels of different host Rab GTPases in L. donovani infected and uninfected macrophages, cells were lysed at indicated time points and Western blot analysis was carried out using specific human antibodies as mentioned in Materials & Methods. Actin was used as loading control. Right panel indicates the quantitation of the respective host Rab proteins. b, Levels of different host Rabs in L. donovani infected and uninfected differentiated macrophages at respective time points were determined by qPCR as described in Materials & Methods. The respective gene amplification was normalized using 18s rRNA as an internal control. All results are represented as mean ± S.D. of three independent experiments and normalized to uninfected control of respective Rabs arbitrarily chosen as one unit. Results of the indicated groups were analyzed by paired t test and levels of significance are indicated by P value. c, Levels of host mRNA of Rab18 in L. donovani infected and uninfected macrophages by limited dilution semi-quantitative RT-PCR. 18s rRNA was used as a control. d, Differentiated THP-1 macrophages were infected with L. donovani and recruitment of host Rab18 on LAMP1 labelled Ld-PVs were determined by immunostaining after 24 h of infection using specific antibody against human Rab18 (1:50) as described in Materials & Methods. THP-1 cells incubated with dead parasites and latex beads were used as control. e, Kinetics of host Rab18 recruitment on Ld-PVs was determined at indicated times as described in Materials & Methods. Right panel indicates the quantitation of host Rab18 recruitment on Ld-PVs at different time points. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). All results are representative of three independent experiments.</p

<i>Leishmania</i>-containing PV specifically recruits LBs.

a, Differentiated THP-1 human macrophages were infected with L. donovani and recruitment of Bodipy 493/503 stained LBs (Green) by LAMP1 stained Ld-PVs (Red) were detected by confocal microscopy after 24 h of infection as described in Materials & Methods. Uninfected cells and cells incubated with latex beads or dead parasite were used as control. b, Recruitment of LipidTOX labelled LBs (Red) by LAMP1 stained Ld-PVs (Green) were determined after 24 h of infection as mentioned in Materials & Methods. Infection with dead parasites was used as control. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). c, To determine the recruitment of Perilipin by LAMP1 stained Ld-PVs (Red), Perilipin was immunostained with specific antibody (Green) in L. donovani infected and uninfected macrophages as described in Materials & Methods. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). d, Percentage of recruitment of different LBs on PVs containing live or fixed parasites were analyzed. All results are represented as mean ± S.D. of three independent experiments. Results of the indicated groups were analyzed by paired t test and levels of significance are indicated by P value. e, Determination of the number and size of LBs in L. donovani infected and uninfected macrophages were determined using appropriate software as described in Materials & Methods. Results are representative of mean±SE of 50 macrophages. f, Recruitment of ER membrane marker on LB and Ld-PV in L. donovani infected macrophages were determined by immunostaining the cells with calnexin specific antibody at indicated time points and analyzed by confocal microscopy. Uninfected cells and cells infected with dead parasite were used as control. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). All results are representative of three independent experiments.</p

Mechanism of overexpression of host Rab18 by <i>Leishmania</i> in infected macrophages.

a, Modulation of the expression of several host miRNAs in L. donovani infected macrophages was revealed by microarray analysis. Relative expression of miRNAs in infected cells was determined in comparison to uninfected control. The whole microarray data have been submitted in Gene Expression Omnibus database (accession number GSE89529). b, Sequence alignment of 3/-UTR of host Rab18 and TRAPPC9 containing 7-mer target site with the seed region of miR-1914-3p predicted by TagetScan tool. Mutation in the target site of 3/-UTR of Rab18 is highlighted in bold. c, Expression of miR-1914-3p in L. donovani infected and uninfected THP-1 macrophages was determined 12 h post infection using qPCR as described in Materials & Methods. d, miR-1914-3p mediated regulation of Rab18 expression was determined by co-transfecting pmir-GLO chimeric construct containing Rab18 3/-UTR or its mutant with miR-1914-3p (40 nM) or control mimic miR into semiconfluent HeLa cells. Luciferase activity was determined from lysed cells after 48 h of transfection as described in Materials & Methods. Results are expressed as relative luciferase activity. Untreated control cells were arbitrarily chosen as one unit. e, THP-1 differentiated macrophages were infected with L. donovani and expression level of Dicer in infected cells was determined at indicated time points using specific antibody against human Dicer. Uninfected cells were used as control. Actin was used as loading control. Right panel indicates the quantitation of host Dicer level at different time points. f, Expression of Dicer was determined in HeLa cells transfected with plasmid containing Flag tagged Ld-gp63 by Western blot analysis using specific antibody against Dicer. Level of gp63 in untransfected and vector transfected cells were also determined by Western blot analysis using anti-Flag antibody. Actin was used as loading control. g, Differentiated THP-1 macrophages were transfected with control miR or miR-1914-3p at indicated concentrations and expression of Rab18 was measured by qPCR as described in Materials & Methods. h, Levels of host Rab18 and Rab5 proteins in control miR or miR-1914-3p (10 nM or 20 nM) transfected THP-1 cells was determined by Western blot analysis using specific antibodies against human Rab18 and Rab5. Actin was used as loading control. i, Similarly, level of TRAPPC9 protein in control miR or miR-1914-3p (10 nM or 20 nM) transfected THP-1 cells was determined by Western blot analysis using human anti-TRAPPC9 antibody. Actin was used as loading control. All results represented as mean ± S.D. of three independent experiments. Results of the indicated groups were analyzed by paired t test and levels of significance are indicated by P value.</p

Shows the inhibition of Rab18 expression by the overexpression of specific siRNA in transfected macrophages.

Shows the inhibition of Rab18 expression by the overexpression of specific siRNA in transfected macrophages.</p

<i>Leishmania</i> acquire fatty acids from LBs for their proliferation in macrophages.

a, Differentiated THP-1 macrophages were incubated with BODIPY-FL-C12 to label the fatty acids in LBs and subsequently cells were infected with L. donovani as described in Methods. Infected cells prelabeled with BODIPY-FL-C12 were chased for indicated time points and appearance of BODIPY-FL-C12 (Green) from LBs in Ld-PVs (Red) was determined. Cells were immunostained with LAMP-1 specific antibody to mark the PVs. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). Uninfected cells were used as control. Right panel shows the quantitation of Ld-PVs acquiring BODIPY-FL-C12 fatty acids in a time dependent way. b, Differentiated THP-1 macrophages were transfected with Rab18 specific siRNA, control siRNA or miR-1914-3p followed by infection with Leishmania. Lipid bodies were stained using Bodipy 493/503 as described above. Cells were fixed and immunostained with LAMP-1 to label the Ld-PVs. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). Right panel shows the quantitation of Ld-PVs acquiring Bodipy 493/503 stained LBs under indicated conditions. All results are representative of three independent experiments. c, Similarly, miR-1914-3p or Rab18 specific siRNA transfected macrophages were infected with L. donovani promastigotes and parasite load in the infected macrophages was microscopically estimated at indicated time. Leishmania and macrophage nuclei were stained with propidium iodide (Red). Results are expressed in the right panel as number of parasites present in 100 macrophages ± S.D. from three independent experiments. Results of control (*) and Rab18 siRNA/miR-1914-3p overexpressed cells were analyzed by paired t test and levels of significance are indicated by P value. d, Similarly, miR-1914-3p transfected and L. donovani infected macrophages were incubated in the presence or absence of fatty acids supplements. Parasite load in the infected macrophages was estimated at 96 h post infection. Untransfected infected cells were used as control. Results are expressed in the right panel as number of parasites present in 100 macrophages ± S.D. from three independent experiments. Results were analyzed by paired t test and levels of significance are indicated by P value.</p

Expression and recruitment of host TRAPPC9 in <i>L</i>. <i>donovani</i> infected macrophages.

a, To determine the levels of host TRAPPC9 and TRAPPC10 in L. donovani infected and uninfected macrophages, cells were lysed at indicated time points and Western blot analysis was carried out using specific human antibodies as mentioned in Materials & Methods. Actin was used as loading control. Lower panel indicates the levels of the host Trappc9 and Trappc10 proteins at indicated time points. b, Levels of TRAPPC9 and TRAPPC10 in infected and uninfected differentiated macrophages at respective time points were determined by qPCR as described in Materials & Methods. The respective gene amplification was normalized using 18s rRNA as an internal control. All results are represented as mean ± S.D. of three independent experiments and normalized to uninfected control of respective Rabs arbitrarily chosen as one unit. Results of the indicated groups were analyzed by paired t test and levels of significance are indicated by P value. c, Differentiated THP-1 macrophages were infected with L. donovani and recruitment of TRAPPC9 on LAMP1 labelled Ld-PVs was determined by immunostaining after 24 h of infection using TRAPPC9 specific antibody. Leishmania and macrophage nuclei were stained with DRAQ5 (Blue). All results are representative of three independent experiments.</p

Demonstrates the recruitment of host Rab18 and LBs stained with LipidTOX on Ld-PVs in <i>L</i>. <i>donovani</i> infected and uninfected macrophages.

Demonstrates the recruitment of host Rab18 and LBs stained with LipidTOX on Ld-PVs in L. donovani infected and uninfected macrophages.</p