Supplementation of nano-selenium (SeNPs) improved growth, immunity, antioxidant enzyme activity, and selenium retention in broiler chicken during summer season.

The present experiment was aimed at finding the optimal supplemental dose of nano-selenium in broiler chicken during the summer season for better performance in terms of growth, blood metabolites, immune response, antioxidant status, and selenium concentration in vital organs. Three-hundred-day-old Vencobb broiler chicks were randomly distributed into five dietary treatment groups with six replicates of 10 chicks each. The dietary treatments were as follows: T1 (control group), basal diet; T2, basal diet with 0.0375 ppm of nano-Se; T3, basal diet with 0.075 ppm of nano-Se; T4, basal diet with 0.15 ppm of nano-Se; T5, basal diet with 0.3 ppm of nano-Se. The experiment was carried out for 35 days. The average gain and feed conversion ratio were best observed in T4 and T5. The antibody titres were significantly higher (P < 0.05) in the treated birds. At the 5th week, erythrocytic glutathione peroxidase, catalase, and superoxide dismutase activities were significantly (P < 0.05) higher and lipid peroxidation values were significantly (P < 0.05) lower in all the nano-Se-treated groups. The Se levels in the liver, breast muscle, kidney, brain, and gizzard were significantly (P < 0.05) increased with increased dietary nano-Se. Histological studies of the liver and kidney in the highest nano-Se-treated groups (T4 and T5) did not show any abnormal changes. It is concluded that supplementation of nano-selenium at 0.15 ppm over and above the basal level improved the performance and protect the birds from summer stress without any adverse effect on the vital organs of chicken.

Knowledge, Attitude and Level of Involvement of Married Males in Family Planning.

Background There is an age-old notion that family planning is women's responsibility disregarding the fact that men have equal responsibility in fertility regulation. Although male involvement is getting more recognition, studies on men's role in family planning are very few in the number in this part of the world. Objective To assess the knowledge, attitude and level of male involvement in family planning and to find out the factors associated with male involvement by contraceptive usage. Method A community based cross-sectional study was done from May to July 2021 among 165 currently married male, who had at least one child, living in Singur district of West Bengal. Cluster sampling method was done to select study participants and data were collected by pre-designed pretested questionnaire. Descriptive statistics, multivariable logistic regression was applied and data were analysed applying SPSS software. Result Only 36.4% participants were directly involved in family planning either by using condom or by withdrawal method but 65.5% participants were indirectly involved in family planning through spousal communication either by approving contraceptive use to their spouse or by decision making regarding family planning. Moreover, barrier of contraceptives usage were side effect (27%) and fear of impotence (25.5%). Male involvement was significantly associated with participant's education [AOR (95% CI= 3.63 (1.45-9.05)], caste [AOR (95% CI= 7.06 (2.55-19.51)], number of living children [AOR (95%CI= 5.01(1.95-12.87)], desire for more child [AOR (95% CI=0.34 (.13-.87)] and attitude on family planning [AOR (95% CI= 3.55 (1.41-8.94)]. Conclusion This study identified the prevailing gender norms in rural areas. Advocacy for male involvement in family planning by health personnel during counselling of eligible couples should help in increasing contraceptive coverage in the long run.

Chemical chaperones reverse early suppression of regulatory circuits during unfolded protein response in B cells from common variable immunodeficiency patients.

B cells orchestrate pro-survival and pro-apoptotic inputs during unfolded protein response (UPR) to translate, fold, sort, secrete and recycle immunoglobulins. In common variable immunodeficiency (CVID) patients, activated B cells are predisposed to an overload of abnormally processed, misfolded immunoglobulins. Using highly accurate transcript measurements, we show that expression of UPR genes and immunoglobulin chains differs qualitatively and quantitatively during the first 4 h of chemically induced UPR in B cells from CVID patients and a healthy subject. We tested thapsigargin or tunicamycin as stressors and 4-phenylbutyrate, dimethyl sulfoxide and tauroursodeoxycholic acid as chemical chaperones. We found an early and robust decrease of the UPR upon endoplasmic reticulum (ER) stress in CVID patient cells compared to the healthy control consistent with the disease phenotype. The chemical chaperones increased the UPR in the CVID patient cells in response to the stressors, suggesting that misfolded immunoglobulins were stabilized. We suggest that the AMP-dependent transcription factor alpha branch of the UPR is disturbed in CVID patients, underlying the observed expression behavior.

Potentially toxic elements in lignite and its combustion residues from a power plant.

The presence of potentially toxic elements in lignite and coal is a matter of global concern during energy extraction from them. Accordingly, Barsingsar lignite from Rajasthan (India), a newly identified and currently exploited commercial source of energy, was evaluated for the presence of these elements and their fate during its combustion. Mobility of these elements in Barsingsar lignite and its ashes from a power plant (Bikaner-Nagaur region of Thar Desert, India) is presented in this paper. Kaolinite, quartz, and gypsum are the main minerals in lignite. Both the fly ash and bottom ash of lignite belong to class-F with SiO2 > Al2O3 > CaO > MgO. Both the ashes contain quartz, mullite, anhydrite, and albite. As, In, and Sr have higher concentration in the feed than the ashes. Compared to the feed lignite, Ba, Co, U, Cu, Cd, and Ni are enriched (10-5 times) in fly ash and Co, Pb, Li, Ga, Cd, and U in bottom ash (9-5 times). Earth crust-normalization pattern showed enrichment of Ga, U, B, Ag, Cd, and Se in the lignite; Li, Ba, Ga, B, Cu, Ag, Cd, Hg, Pb, and Se, in fly ash; and Li, Sr, Ga, U, B, Cu, Ag, Cd, Pb, and Se in bottom ash. Hg, Ag, Zn, Ni, Ba, and Se are possibly associated with pyrite. Leaching test by toxicity characteristic leaching procedure (TCLP) showed that except B all the elements are within the safe limits prescribed by Indian Standards.

Growth associated polyhydroxybutyrate production by the novel Zobellellae tiwanensis strain DD5 from banana peels under submerged fermentation.

In view of environmental pollution by fossil fuel-based plastics, it has become imperative to find out an alternative biodegradable plastic for sustainability. In this context, polyhydroxy butyrate (PHB) production was carried out by the Zobellella sp. DD5 using inexpensive banana peels as the carbon source. Under optimized condition, 1.13 g/L (47.3%) of PHB was produced by the bacteria in growth associated mechanism. The CO group of PHB was detected from the high intense absorption band (1719 cm-1) of FTIR spectroscopic analysis. NMR and GC-MS results are also identical with the chemical shift signal CH, CH2 and CH3 group of PHB. The PHB is crystalline in nature and degree of crystallinity (Xc) - 34.38%, melting temperature (Tm) - 169 °C, thermal decomposition temperature (Td) - 248 °C as detected by XRD and DTA respectively. Rough surface morphology of PHB film was validated by AFM and SEM imaging that improves biodegradability of the PHB. The Young's modulus, tensile strength and elongation at break depicted hard and brittle nature of PHB. Fluorescence-activated cell sorting (FACS) confirmed cytocompatibility of PHB at 500 µg/mL in human embryonic kidney (HEK-293) cell line. The non-cytotoxic PHB can be used for various biomedical and agricultural applications in future.

Comparative analysis of PHAs production by Bacillus megaterium OUAT 016 under submerged and solid-state fermentation.

In view of risk coupled with synthetic polymer waste, there is an imperative need to explore biodegradable polymer. On account of that, six PHAs producing bacteria were isolated from mangrove forest and affilated to the genera Bacillus & Pseudomonas from morpho-physiological characterizations. Among which the potent PHAs producer was identified as Bacillus megaterium OUAT 016 by 16S rDNA sequencing and in-silico analysis. This research addressed a comparative account on PHAs production by submerged and solid-state fermentation pertaining to different downstream processing. Here, we established higher PHAs production by solid-state fermentation through sonication and mono-solvent extraction. Using modified MSM media under optimized conditions, 49.5% & 57.7% of PHAs were produced in submerged and 34.1% & 62.0% in solid-state fermentation process. Extracted PHAs was identified as a valuable polymer PHB-co-PHV and its crystallinity & thermostability nature was validated by FTIR, 1H NMR and XRD. The melting (Tm) and thermal degradation temperature (Td) of PHB-co-PHV was 166 °C and 273 °C as depicted from DTA. Moreover, FE-SEM and SPM surface imaging indicated biodegradable nature, while FACS assay confirmed cytocompatibility of PHB-co-PHV.

Selective activation of transcription by a novel CCAAT binding factor.

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the alpha 2(I) collagen, the alpha 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

Role of the CCAAT-binding protein CBF/NF-Y in transcription.

The CCAAT motif is one of the common promoter elements present in the proximal promoter of numerous mammalian genes transcribed by RNA polymerase II. CBF (also called NF-Y and CP1) consists of three different subunits and interacts specifically with the CCAAT motif. In each CBF subunit, the segment needed for formation of the CBF-DNA complex is conserved from yeast to human and, interestingly, the conserved segment of two CBF subunits, CBF-A and CBF-C, are homologous to the histone-fold motif of eukaryotic histones and archaebacterial histone-like protein HMf-2. The histone fold motifs of CBF-A and CBF-C interact with each other to form a heterodimer that associates with CBF-B to form a heterotrimeric CBF molecule, which then binds to DNA.

Multi-element detection in sea water using preconcentration procedure and EDXRF technique.

A method was optimized for detecting trace elements in sea water using Energy dispersive X-ray fluorescence (EDXRF) technique. Sea water samples were pre-concentrated using ammonium pyrrolidinedithiocarbamate (APDC) as chelating agent and methyl isobutyl ketone (MIBK) as organic phase. The preconcentrated samples were dried to form thin films on mylar substrate and analysed using EDXRF spectrometer. The multi-element standard samples prepared in synthetic sea water were used for EDXRF instrument calibration. The instrument was calibrated for 11 elements namely As, Cd, Co, Cr, Fe, Ga, Pb, Se, Sc, V and Zn using linear regression method for concentration up to 200 ppb. The detection limits achieved for As, Cd, Co, Cr, Fe, Ga, Pb, Se, Sc, V and Zn were 13, 70, 5.1, 36, 15, 36, 23, 11, 20, 13 and 40 ppb respectively. The optimized method was used for determination of elements in sea water collected from the Thane creek, Mumbai, India. The results were checked for accuracy by comparing it with inductively coupled plasma atomic emission spectroscopy (ICP AES) technique. The comparison showed the discrepancy of results to be insignificant at 95% confidence level.

Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule.

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

Transcriptional scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the class II major histocompatibility complex promoter.

Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.

Three classes of mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex.

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.

An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.

Adsorption of zinc from aqueous solution using chemically treated newspaper pulp.

Adsorption of zinc was studied using chemically modified newspaper pulp as an adsorbent in the aqueous medium. Quantitative chemical analysis showed the presence of trace quantities of some inorganic elements along with phosphorous in TNP. The experimental adsorption data fitted reasonably well to both Freundlich and Langmuir isotherm. pHzpc of TNP was 5.1, which indicated that the adsorbent was more potential for cationic adsorption. The adsorption kinetic data followed a pseudo-second order model for zinc. Optimum Zn(2+) loading was 9.20 mg/g for 10.31 mg/l initial zinc concentration at pH 5.80. Zn(2+) loading on TNP was dependent on initial zinc concentration. TNP was a potential adsorbent for the removal of Zn from the effluent of electroplating industry.

Flow investigation in an industrial-scale soaker using radiotracer technique.

Flow dynamics of heavy petroleum residue in an industrial-scale soaker operating in a petroleum refinery was investigated. Residence time distributions (RTDs) of the residue were measured using radiotracer technique. Bromine-82 as dibromobiphenyl was used as radiotracer for tracing the petroleum residue. The measured RTDs were treated and mean residence times (MRTs) were determined. The measured RTD data was simulated using a combined model i.e. axial dispersion model in parallel with tanks-in-series with stagnant volume and exchange. The results of the model simulation fitted very well to the experimentally measured data and identified bypassing or existence two parallel flow paths within the soaker.

Bioconversion of fish solid waste into PHB using Bacillus subtilis based submerged fermentation process.

Currently, one of the major problem affecting the world is solid waste management, predominantly petroleum-based plastic and fish solid waste (FSW). However, it is very difficult to reduce the consumption of plastic as well as fish products, but it is promising to convert FSW to biopolymer to reduce eco-pollution. On account of that, the bioconversion of FSW extract to polyhydroxybutyrate (PHB) was undertaken by using Bacillus subtilis (KP172548). Under optimized conditions, 1.62 g/L of PHB has been produced by the bacterium. The purified compound was further characterized by advanced analytical technologies to elucidate its chemical structure. Results indicated that the biopolymer was found to be PHB, the most common homopolymer of polyhydroxyalkanoates (PHAs). This is the first report demonstrating the efficacy of B. subtilis to utilize FSW extract to produce biopolymer. The biocompatibility of the PHB against murine macrophage cell line RAW264.7 demonstrated that, it was comparatively less toxic, favourable for surface attachment and proliferation in comparison with poly-lactic acid (PLA) and commercially available PHB. Thus, further exploration is highly indispensable to use FSW extract as a substrate for production of PHB at pilot scale.

Protection from experimental colitis by theaflavin-3,3'-digallate correlates with inhibition of IKK and NF-kappaB activation.

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) is associated with activation of nuclear factor kappa B (NF-kappaB) involved in regulating the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokine genes. As theaflavin-3,3'-digallate (TFDG), the most potent anti-oxidant polyphenol of black tea, down-regulates NF-kappaB activation, we investigated if TFDG is beneficial in colonic inflammation by suppressing iNOS and proinflammatory cytokines. EXPERIMENTAL APPROACH: The in vivo efficacy of TFDG was assessed in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Both mRNA and protein levels of proinflammatory cytokines and iNOS were analyzed in colon tissue treated with or without TFDG. NF-kappaB activation was determined by electrophoretic mobility shift assay and levels of NF-kappaB inhibitory protein (IkappaBalpha) were analyzed by Western blotting. KEY RESULTS: Oral administration of TFDG (5 mg kg(-1) daily i.g.) significantly improved TNBS-induced colitis associated with decreased mRNA and protein levels of TNF-alpha, IL-12, IFN-gamma and iNOS in colonic mucosa. DNA binding and Western blotting revealed increase in NF-kappaB activation and IkappaBalpha depletion in TNBS-treated mice from Day 2 through Day 8 with a maximum at Day 4, which resulted from increased phosphorylation of IkappaBalpha and higher activity of IkappaB kinase (IKK). Pretreatment with TFDG markedly inhibited TNBS-induced increases in nuclear localization of NF-kappaB, cytosolic IKK activity and preserved IkappaBalpha in colon tissue. CONCLUSIONS AND IMPLICATIONS: TFDG exerts protective effects in experimental colitis and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.

Inhibition of FOXC2 restores epithelial phenotype and drug sensitivity in prostate cancer cells with stem-cell properties.

Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties.

A study on arsenic adsorption on polymetallic sea nodule in aqueous medium.

A detailed study on As(III) and As(V) adsorption on polymetallic sea nodule in aqueous medium has been reported. Elemental composition of sea nodule comprises primarily, iron, manganese and silicon with trace quantities of aluminium, copper, cobalt and nickel. As(V) adsorption on sea nodule is dependent on pH while As(III) is insensitive to it. Adsorption data broadly follow Langmuir isotherm. Kinetic data follow a pseudo-second-order model for both As(III) and As(V). Arsenic loading on sea nodule is dependent on initial arsenic concentration. Optimum As(III) loading is 0.74 mg/g at 0.34 mg/L and for As(V) it is 0.74 mg/g at 0.78mg/L. As(III) adsorption is broadly independent of ionic environment. Except for PO(4)(3-), As(III) adsorption is not influenced by anions but cations influence it significantly. As(V) adsorption, on the other hand, is influenced by anions and not by cations. Experimental evidence indicates an inner sphere complex for As(III) and partial inner and partial outer sphere complex for As(V). Both As(III) and As(V) adsorptions are marked with very little desorption in the pH range of 2-10. Sea nodule can speciate As(III) and As(V) in groundwater at or above pH 6. Sea nodule was successfully tested as an adsorbent for the removal of arsenic from six contaminated groundwater samples of West Bengal, India, containing arsenic in the range 0.04-0.18mg/L.