Effects of driver work-rest patterns, lifestyle and payment incentives on long-haul truck driver sleepiness

The aim of the study is to identify and model the role of payment incentives, driver work-rest patterns and other lifestyle habits influencing the drowsy driving behavior among long-haul truck drivers. To achieve this aim, this study targeted two main objectives: (1) to examine the significant differences between the groups of drowsy and non-drowsy drivers based on the opportunities of monetary incentives and (2) to examine the role of different factors: driver demographics, work-rest patterns, lifestyle and occupational characteristics particularly incentives associated with driving in causing driver sleepiness among Indian truck drivers. The study is based on interview responses from 453 long-haul truck drivers approached in three Indian cities- Mumbai, Indore and Nagpur. Initial principal component analysis of the responses related to financial incentives (occupational characteristics) resulted into two correlated factors: (i) willingness to earn extra payments if offered (WEP) and (ii) incentives available in the current driving experience (ICD) that influence driver work-rest patterns and alertness while driving. Kruskal-Wallis test showed a significant difference among the groups of sleepy and non-sleepy drivers due to these factors (WEP and ICD). Finally, a logistic regression model showed that long driving duration, working days per week, rest patterns, insufficient sleeping hours and history of violations were found significantly associated with drowsy driving among the long-haul truck drivers. Increase in consumption of caffeine and tobacco indicated reduction in driver alertness. According to the model results, the odds of drowsy driving were 77% less for drivers between 46-55 years compared to the young drivers (<25 years). Driving under the influence of financial incentives was observed to increase the odds of falling asleep by 1.58 times among the truck drivers. This was apparently the most interesting and intriguing result of the study indicating the need for further research on the influence of financial or socio-economic motivations to sleepiness

Compensatory Role of Inositol 5-Phosphatase INPP5B to OCRL in Primary Cilia Formation in Oculocerebrorenal Syndrome of Lowe

<div><p>Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in <i>OCRL</i>, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. <i>INPP5B</i> is a paralog of <i>OCRL</i> and shares similar structural domains. The roles of <i>OCRL</i> and <i>INPP5B</i> in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm’s canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of <i>INPP5B</i>. This is further supported by the defective cilia formation in zebrafish Kupffer’s vesicles and in cilia-dependent melanosome transport assays in <i>inpp5b</i> morphants. Taken together, this study indicates that <i>OCRL</i> and <i>INPP5B</i> are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.</p></div

Effect of INPP5B CAAX mutant on cilia localization.

<p>(A) Domain structure of human INPP5B protein. (B) hTERT-RPE1 cells were transduced by <i>GFP-Inpp5b</i> or <i>GFP-Inpp5bΔCAAX</i> lentivirus, starved for 48 hr, and then analyzed by immunostaining with anti-acetylated alpha-tubulin antibody. Scale bar 10 micron. (C) Lengths of primary cilia in <i>Inpp5b</i> and <i>Inpp5b</i>-<i>delta</i>-CAAX cells. hTERT-RPE1 cells were transduced with either <i>GFP-Inpp5b</i> or <i>GFP-Inpp5b</i>-<i>delta</i>-<i>CAAX</i> lentivirus, serum starved for 48 hr, and stained with anti-acetylated alpha-tubulin antibody. Scatter plot showing distribution pattern of ciliary length (3.4±0.4 micron in <i>INPP5B</i> and 2.8±0.3 micron in <i>Inpp5bΔCAAX</i> cells, unpaired t-test, p = 1.36E-06, n >160 cilia, three independent experiments). (D) <i>INPP5BΔCAAX</i> mRNA failed to rescue the loss of <i>inpp5b</i>. KV cilia of zebrafish embryos injected with <i>inpp5b</i> MO (4 ng), <i>inpp5b</i> MO (4 ng) with <i>INPP5B</i> WT mRNA (500 ng) and <i>inpp5b</i> MO (4 ng) with <i>INPP5B ΔCAAX</i> mRNA (500 ng) at 6-somite stage were immunostained with acetylated α-tubulin (red), representative images are shown (dash line indicates border of KV). Scale bar 10 micron. (E–F) Quantification of length (E) and number (F) of KV cilia in zebrafish embryos injected with <i>inpp5b</i> MO (4 ng), <i>inpp5b</i> MO (4 ng) with <i>INPP5B</i> WT mRNA (500 ng) and <i>inpp5b</i> MO (4 ng) with <i>INPP5B ΔCAAX</i> mRNA (500 ng). (N >20 embryos, three independent experiments, unpaired t-test, * p = 3.9E-26 in E and * p = 9E-06 in F).</p

INPP5B can partly rescue the defect of OCRL in primary cilia development.

<p>(A) INPP5B protein levels in HEK293T cells. <i>GFP-Inpp5b</i> lentivirus was generated in HEK293T cells; both GFP-Inpp5b and endogenous INPP5B were immunoblotted in 40 microgram lysates; beta-actin levels are shown. (B) NHF 558, Lowe 1676, and Lowe 3265 fibroblasts were transduced with control, <i>Inpp5b</i> or <i>Inpp5b ΔCAAX</i> lentivirus, serum-starved for 48-hours, and immunostained with acetylated alpha -tubulin. Quantification of cilia length is shown (n >100 cilia, three independent experiments, unpaired t-test, * p = 0.004, ** p = 0.006). (C) <i>Inpp5b</i> mRNA partly rescued the loss of <i>Ocrl</i>. KV cilia of zebrafish embryos injected with <i>ocrl</i> MO (4 ng) with <i>Inpp5b</i> WT mRNA (500 ng) and <i>ocrl</i> MO (4 ng) with <i>Inpp5bΔCAAX</i> mRNA (500 ng) at 6-somite stage were immunostained with acetylated α-tubulin (red), representative images are shown (dash line indicates border of KV). Scale bar 10 micron. (D) Quantification of length of KV cilia in zebrafish embryos injected with <i>ocrl</i> MO (4 ng) with <i>Inpp5b</i> WT mRNA (500 ng) and <i>ocrl</i> MO (4 ng) with <i>Inpp5bΔCAAX</i> mRNA (500 ng). (N >20 embryos, three independent experiments, unpaired t-test, * p = 2.2E-04).</p

INPP5B and OCRL act synergistically in eye development.

<p>(A) Representative phenotypes of morphants co-injected with <i>ocrl</i> and <i>inpp5b</i> MO. Scale bar 250 micron. (B) Dose-dependent effect of two morpholinos in zebrafish. Control <i>ocrl</i> and <i>inpp5b</i> MO at indicated doses were injected into zebrafish embryos, and different grades of phenotypes were quantified at 48 hpf (N >250 embryos per group). (C) Transgenic <i>mOcrl^−/−:mInpp5b^−/−:hINPP5B</i>^+/+ mouse eyes were sectioned and stained by H&E, anti-OCRL antibody (green) or anti-INPP5B antibody (green), and DAPI (blue). Staining of trabecular meshwork cells in vesicular pattern (arrow), dash line indicates border of cornea and iris. Scale bar 10 micron. (D) Photoreceptor cells (arrow) from the transgenic mouse eye section stained with H&E, anti-OCRL antibody (green) or anti-INPP5B antibody (green), and DAPI (blue). Scale bar 10 micron. (E) Lens epithelial cells (arrow) stained with H&E, anti-OCRL antibody (green) or anti-INPP5B antibody (green), and DAPI (blue). Scale bar 10 micron.</p

INPP5B localizes to primary cilia.

<p>(A) hTERT-RPE1 cells were starved for 48 hr, and analyzed by immunostaining with anti-INPP5B antibody and anti-acetylated alpha-tubulin antibody. Scale bar 5 micron. (B) Control and <i>INPP5B</i> shRNA hTERT-RPE1 cells were starved for 48 hr, and analyzed by immunostaining with anti-INPP5B antibody and anti-acetylated alpha-tubulin antibody. Scale bar 5 micron. (C-D) Control and <i>INPP5B</i> shRNA hTERT-RPE1 cells were starved for 24 hr or 48 hr, and analyzed by immunostaining with anti-acetylated alpha-tubulin antibody. Percent ciliated cells (C) and the length of cilia (D) were quantified. (n = the number of cilia n >100 cilia, three independent experiments, unpaired t-test, * p = 2.1E-08 in D, * p = 3.1 E-09 in E; ns, not statistically significant).</p

<i>Inpp5b</i> morpholino affects multiple organ development in zebrafish.

<p>(A) Immunoblot analysis of 40 microgram of total lysates of zebrafish embryo injected with control MO (4 ng) or <i>inpp5b</i> MO (4 ng) at 48 hpf with anti-INPP5B and anti-beta-actin antibodies. (B) Zebrafish embryos were injected with <i>p53</i> MO (2 ng) or <i>p53</i> MO (2 ng) and <i>inpp5b</i> MO (4 ng). Representative phenotypes of microphthalmia (black arrowhead), pericardial edema (small arrow), body axis asymmetry, kinked tail (white arrow), pronephric cyst formation (red arrow), and hypopigmentation were observed at 48 hpf. Scale bar 250 micron. (C) Dose-dependent effect of morpholinos in zebrafish. Control or <i>inpp5b</i> MO at indicated doses was injected into zebrafish embryos, and phenotypes of microphthalmia, kinked tail, and body asymmetry were quantified at 48 hpf (ANOVA, F = 92, p = 3.6E-10), kinked tail (ANOVA, F = 3.6, p = 0.08), and body asymmetry (ANOVA, F = 5.2, p = 0.04). (N = the number of injected embryos N >50). (D) Quantification of eye size of morphants at 24 hpf, 48 hpf and 72 hpf. The eye size was determined by the longest diameters in dorsal view. (N >40 embryos, three independent experiments, unpaired t-test, * p = 4.98E-07 ** p = 2.1E-10, ns, not statistically significant). (E) Cresyl violet staining of ocular sections of zebrafish larvae (5 dpf) injected with control MO (4 ng) or <i>inpp5b</i> MO (4 ng). Scale bar 30 micron. (F) Zebrafish embryos were injected with control MO (4 ng), <i>inpp5b</i> MO (4 ng) or <i>inpp5b</i> MO (4 ng) and <i>Inpp5b</i> WT mRNA (<i>Inpp5b</i> mRNA, 500 pg). Representative phenotypes of microphthalmia (arrowhead), pericardial edema (arrow), body axis asymmetry, kinked tail (white arrow), pronephric cyst formation (red arrow), and hypopigmentation were observed at 48 hpf. Scale bar 250 micron. (G) Quantification of eye size of zebrafish morphants at 24 hpf and 48 hpf. (N >40 embryos, three independent experiments, unpaired t-test, * p = 4.5E-05). (H) The ventral sides of embryos were injected with control MO (4 ng), <i>inpp5b</i> MO (4 ng) or <i>inpp5b</i> MO (4 ng) and <i>Inpp5b</i> WT mRNA (500 pg). Scale bar 100 micron.</p

Defects of cilia formation in <i>Inpp5b</i> zebrafish morphants.

<p>(A) <i>INPP5B</i> WT mRNA rescue the loss of <i>inpp5b</i>. KV cilia of zebrafish embryos injected with control MO (4 ng), <i>inpp5b</i> MO (4 ng) or <i>inpp5b</i> MO (4 ng) and <i>INPP5B</i> WT mRNA (500 ng) at 6-somite stage were immunostained with acetylated α-tubulin (red), representative images are shown (dash line indicates border of KV). Scale bar 10 micron. (B–C) Quantification of number (B) and length (C) of KV cilia in zebrafish embryos injected with control MO (4 ng), <i>inpp5b</i> MO (4 ng) or <i>inpp5b</i> MO (4 ng) and <i>INPP5B</i> WT mRNA (500 ng). (N >20 embryos, three independent experiments, unpaired t-test, * p = 3.4E-03 in B and * p = 1.9E-23 in C). (D) <i>INPP5B</i> WT mRNA rescue of <i>inpp5b</i> pronephric cilia formation. Representative image of pronephric cilia of zebrafish embryos at 24 hpf stage, injected with control MO (4 ng), <i>inpp5b</i> MO (4 ng) or <i>inpp5b</i> MO (4 ng) and <i>INPP5B</i> WT mRNA (500 ng), immunostaining with acetylated α-tubulin (red). Scale bar 10 micron. (E) Pronephric cilia length of control and <i>inpp5b</i> MO. Pronephric cilia of zebrafish embryos injected with control MO (4 ng), <i>inpp5b</i> MO (4 ng) or <i>inpp5b</i> MO (4 ng) and <i>INPP5B</i> WT mRNA (500 ng) at 24 hpf stage were analyzed by immunostaining with acetylated alpha-tubulin and cilia length was measured. (N >200 cilia; three independent experiments, unpaired t-test, * p = 5.45E-18). (F) <i>Ocrl</i> and <i>Inpp5b</i> morphants showed slowed retrograde melanosome transport. Representative photos are shown for the melanosomes in <i>Ocrl</i> and <i>Inpp5b</i> morphants before and after treatment with epinephrine in 5 dpf embryos (box, region of pigment evaluation). (G) Quantification of the response time for epinephrine treatments in the control MO (2 ng), <i>ocrl</i> MO (2 ng), <i>ocrl</i> MO (2 ng) and <i>OCRL</i> WT mRNA (500 pg), <i>inpp5b</i> MO (2 ng), and <i>inpp5b</i> MO (2 ng) and <i>Inpp5b</i> WT mRNA (500 pg) embryos (N >30 embryos, three independent experiments, unpaired t-test, * p = 4.6E-50, ** p = 2.3E-43).</p

Localization of OCRL and INPP5B in human ocular tissue.

<p>(A) Diagram of trabecular meshwork and Schlemm’s canal endothelial cells (aqueous flow, red; trabecular meshwork, arrow; Schlemm’s canal endothelial cells, arrowhead). (B) Human eye sectioned and stained by H&E or immunofluorescence with anti-OCRL (green) or anti-INPP5B antibody (green), and DAPI (blue). Staining of trabecular meshwork cells in vesicular pattern (insert, arrow) and Schlemm’s canal (arrowhead). Scale bar 10 micron. (C) Lens epithelial cells (arrow) stained with H&E, anti-OCRL antibody (green) or anti-INPP5B antibody (green), and DAPI (blue). Scale bar 10 micron. (D) Ciliary body epithelial (arrowhead) cells stained with H&E, anti-OCRL antibody (green) or anti-INPP5B antibody (green), and DAPI (blue). Scale bar 10 micron. (E) Mouse eye sectioned and stained with H&E, anti-OCRL antibody (green) or anti-INPP5B antibody (green), and DAPI (blue). Staining of trabecular meshwork cells in vesicular pattern (dash line indicates border of trabecular meshwork). Scale bar 10 micron. (F) Immunoblot of <i>INPP5B</i> expression in 30 microgram lysates of hTERT-RPE1 shRNA knockdown cells compared to beta-actin. (G) Immunoblot analysis of 40 microgram lysates of human and mouse trabecular meshwork with anti-OCRL, anti-INPP5B and anti-beta-actin antibodies.</p

Surface-Modified Lyotropic Crystalline Nanoconstructs Bearing Doxorubicin and Buparvaquone Target Sigma Receptors through pH-Sensitive Charge Conversion to Improve Breast Cancer Therapy

In the current study, we aimed to develop lyotropic crystalline nanoconstructs (LCNs) based on poly(l-glutamic acid) (PLG) with a two-tier strategy. The first objective was to confer pH-responsive charge conversion properties to facilitate the delivery of both doxorubicin (DOX) and buparvaquone (BPQ) in combination (B + D@LCNs) to harness their synergistic effects. The second goal was to achieve targeted delivery to sigma receptors within the tumor tissues. To achieve this, we designed a pH-responsive charge conversion system using a polymer consisting of poly(ethylenimine), poly(l-lysine), and poly(l-glutamic acid) (PLG), which was then covalently coupled with methoxybenzamide (MBA) for potential sigma receptor targeting. The resulting B + D@LCNs were further modified by surface functionalization with PLG–MBA to confer both sigma receptor targeting and pH-responsive charge conversion properties. Our observations indicated that at physiological pH 7.4, P/B + D-MBA@LCNs exhibited a negative charge, while under acidic conditions (pH 5.5, characteristic of the tumor microenvironment), they acquired a positive charge. The particle size of P/B + D-MBA@LCNs was determined to be 168.23 ± 2.66 nm at pH 7.4 and 201.23 ± 1.46 nm at pH 5.5. The crystalline structure of the LCNs was confirmed through small-angle X-ray scattering (SAXS) diffraction patterns. Receptor-mediated endocytosis, facilitated by P/B + D-MBA@LCNs, was confirmed using confocal laser scanning microscopy and flow cytometry. The P/B + D-MBA@LCNs formulation demonstrated a higher rate of G2/M phase arrest (55.20%) compared to free B + D (37.50%) and induced mitochondrial depolarization (59.39%) to a greater extent than P/B + D@LCNs (45.66%). Pharmacokinetic analysis revealed significantly improved area under the curve (AUC) values for both DOX and BPQ when administered as P/B + D-MBA@LCNs, along with enhanced tumor localization. Tumor regression studies exhibited a substantial reduction in tumor size, with P/B + D-MBA@LCNs leading to 3.2- and 1.27-fold reductions compared to B + D and nontargeted P/B + D@LCNs groups, respectively. In summary, this two-tier strategy demonstrates substantial promise for the delivery of a drug combination through the prototype formulation. It offers a potential chemotherapeutic option by minimizing toxic effects on healthy cells while maximizing therapeutic efficacy