Association between the MTHFR (rs1801133) gene variation and serum trace elements levels (Copper and Zinc) in individuals diagnosed with neural tube defects.

BACKGROUND AND AIMS: Neural tube defects (NTDs) occur when the neural tube fails to close within 28 days of human embryonic development. This results in central nervous system disorders like anencephaly, spina bifida, and encephalocele. Early diagnosis and treatment are crucial to minimize their impact on an individual's health and well-being. The present study aims to define the association between prenatal exposure to trace elements (Cu and Zn) and the single nucleotide polymorphism (SNP) of the MTHFR gene involved in folate metabolism pathways in neural tube defects in children and their mothers. MATERIAL AND METHODS: A cross-sectional study involving 331 participants (90 NTD cases, 88 healthy mothers, 85 NTD children, and 68 healthy children) from antenatal check-ups in Obstetrics and Gynaecology and Pediatric Surgery for Neural Tube Defects in the Outpatient Department (OPD) and Inpatient Department (IPD). Assessed Cu and Zn concentrations and their associations. Genomic DNA was extracted, and real-time PCR was used to determine genotypes. Atomic absorption spectrophotometry measured trace elements. Statistical analyses included Chi-Square tests, odds ratios, and Mann-Whitney U tests. RESULTS: Significant associations were found between MTHFR C677T genotypes and NTD risk in mothers (p = 0.0491) and children (p = 0.0297). Allelic frequency analysis indicated a T allele association with NTD risk in children (p = 0.0107). Recessive models showed significant associations in mothers (p = 0.0169) and children (p = 0.1678). Cu levels differed significantly between NTD cases and controls (p < 0.0001), with MTHFR genotypes influencing Cu levels. Zinc levels also varied significantly (p < 0.0001). CONCLUSION: This study reveals complex associations between MTHFR C677T genotypes, trace element concentrations, and NTD risk in mothers and children. This targeted approach allows healthcare providers to identify at-risk pregnancies early, enabling personalised interventions like folic acid supplementation and counselling to moderate neural tube defect (NTD) risk in a future pregnancy.

BCR-ABL kinase domain mutations in CML patients, experience from a tertiary care center in North India.

Background: Chronic Myeloid Leukemia is characterized by the presence of the Philadelphia Chromosome (Ph) which contains the BCR::ABL1 fusion gene that occurs due to a reciprocal translocation between chromosomes 9 and 22. This accounts for up to 15 % of all adult leukemias [1]. Most patients treated with first line tyrosine kinase inhibitor (TKI) imatinib achieve durable response but may undergo relapse at some stage [2]. The most important mechanism that may confer imatinib resistance is point mutation within BCR::ABL kinase domain. Other generation ABL tyrosine kinase inhibitors such as dasatinib, nilotinib, bosutinib and ponatinib help to overcome imatinib resistance [3]. Sensitivity of the patient to each of the above TKIs depends upon the individual candidate mutation present. Thus, it is important to perform mutation analysis for effective therapeutic management of CML patients once they show imatinib resistance. We used direct sequencing to identify the different types of mutations responsible for resistance of imatinib treatment from north India. Methods: In this study, the patient resistance for the imatinib were analyzed for BCR::ABL kinase domain mutation by direct sequencing and the detected mutations along with their percentage prevalence were reported. Results: 329 patients with CML-CP were analyzed for BCR::ABL kinase domain mutation. Total 66 (20.06 %) patients out of 329 had mutation in at least one of the domains of BCR::ABL conferring resistance to different generations of TKI. Mutations in BCR::ABL kinase domain was observed in different domain of BCR::ABL. ATP binding P-Loop (42.42 %), Direct binding site (36.36 %), C-Loop (10.60 %), A-Loop (6.06 %), SH2 contact (3.03 %), SH3 contact (1.51 %). Conclusion: Total 20.06 % patients (66/329) show mutation in at least one of the structural motifs of BCR-ABL kinase domain, which further confer the resistance to a particular generation of TKI.

In Silico Prediction of Potential Inhibitors for Targeting RNA CAG Repeats via Molecular Docking and Dynamics Simulation: A Drug Discovery Approach.

Spinocerebellar ataxia (SCA) is a rare neurological illness inherited dominantly that causes severe impairment and premature mortality. While each rare disease may affect individuals infrequently, collectively they pose a significant healthcare challenge. It is mainly carried out due to the expansion of RNA triplet (CAG) repeats, although missense or point mutations can also be induced. Unfortunately, there is no cure; only symptomatic treatments are available. To date, SCA has about 48 subtypes, the most common of these being SCA 1, 2, 3, 6, 7, 12, and 17 having CAG repeats. Using molecular docking and molecular dynamics (MD) simulation, this study seeks to investigate effective natural herbal neuroprotective compounds against CAG repeats, which are therapeutically significant in treating SCA. Initially, virtual screening followed by molecular docking was used to estimate the binding affinity of neuroprotective natural compounds toward CAG repeats. The compound with the highest binding affinity, somniferine, was then chosen for MD simulation. The structural stability, interaction mechanism, and conformational dynamics of CAG repeats and somniferine were investigated via MD simulation. The MD study revealed that during the simulation period, the interaction between CAG repeats and somniferine stabilizes and results in fewer conformational variations. This in silico study suggests that Somniferine can be used as a therapeutic medication against RNA CAG repeats in SCA.

The Rapid Evaluation of Down Syndrome With Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR): A Pilot Study Among the Population in Eastern Uttar Pradesh, India.

Background and objective Down syndrome (DS) is characterized by the presence of an additional chromosome; it is a typical chromosomal disorder causing intellectual disability in individuals. The diagnostic process for DS often involves conventional karyotyping, which can be time-consuming. Trisomy 21 and other chromosomal abnormalities may now be quickly and accurately diagnosed using quantitative fluorescence polymerase chain reaction (QF-PCR). In light of this, this study aimed to investigate chromosomal abnormalities in DS using conventional karyotyping and QF-PCR among the population in eastern Uttar Pradesh, India. Methods Blood samples from 40 individuals with clinically diagnosed DS were collected. Conventional karyotyping involved standard cytogenetic techniques, while QF-PCR utilized DNA extraction and analysis with chromosome-specific short tandem repeat (STR) markers. Results Various distinct physical characteristics were observed in the DS individuals, such as mongoloid slant and low-set ears. Karyotyping and QF-PCR analyses revealed different chromosomal configurations associated with DS trisomy 21, with additional chromosomal abnormalities found in some individuals, including partial monosomy 18 and mosaic trisomy 21. However, in a few cases, neither karyotyping nor QF-PCR revealed any abnormalities. Conclusions The study demonstrated that QF-PCR is a reliable and rapid method for diagnosing DS, providing results within 24 hours. This approach allows for the simultaneous diagnosis of a large number of samples and reduces the time required to obtain results. In the diagnostic procedure for DS, we believe QF-PCR will prove to be a useful tool. Furthermore, therapeutic interventions based on their clinical traits and molecular karyotyping can enhance the quality of life of people with DS.

Post-COVID-19 headache- NDPH phenotype: a systematic review of case reports.

Background and objectives: Post-acute COVID-19 syndrome or "long COVID" affects patients even after the recovery from Covid infection in various ways. Persistent headache or New Daily Persistent Headache (NDPH) is one of such symptoms. In this review, we will discuss about the case-reports of post covid-19 headache- NDPH phenotype both after and in the course of COVID-19 infection. Methods: Case reports/studies talked about patients having NDPH around the disease either immediately or late post COVID were included. Data was taken from the source and synthesised on a qualitative basis. Results: Literature search showed 3,538 articles, out of which 12 were screened as per the eligibility criteria and finally, 4 case reports on NDPH and Covid-19 were chosen for analysis from the database and by human search. All case reports justify the criteria for acceptability in quality for this systematic review. Conclusion: NDPH in and around Covid 19 infection is something that is currently an ingenious debated topic in the scientific community. More case studies should be written and published on the same subject so that a large systematic review could be conducted. Trial Registration Information: The review is registered in Prospero with no. Identifier (CRD42022354912). Systematic Review Registration: https://www.crd.york.ac.uk/, PROSPERO (CRD42022354912).

Correction: SARS-CoV-2 Mutations Responsible for Immune Evasion Leading to Breakthrough Infection.

[This corrects the article DOI: 10.7759/cureus.29544.].

SARS-CoV-2 Mutations Responsible for Immune Evasion Leading to Breakthrough Infection.

BACKGROUND AND OBJECTIVES: India had faced a devastating second outbreak of COVID-19 infection, in which a majority of the viral sequences were found to be of the B.1.617.2 lineage (Delta-variant). While India and the world focused on vaccination, reports of vaccine-immunity evasion by the virus, termed "breakthrough cases", emerged worldwide. Our study was focused on the primary objective to identify the mutations associated with breakthrough infections SARS-CoV-2. METHODS: In our study, we extracted the SARS-CoV-2 RNA (ribonucleic acid) from reverse transcription-polymerase chain reaction (RT-PCR) positive COVID-19 patients, and 150 random samples were sent for sequencing to the Centre for Cellular & Molecular Biology, Hyderabad. Whole genome sequences of 150 SARS-CoV-2 viral samples were analyzed thoroughly. We mostly found B.1.617 and its sub-lineages in the genomic sequencing results. RESULTS AND INTERPRETATION: On further analysis of patient data, it was seen that nine patients had been vaccinated against the SARS-CoV-2 previously. These nine patients had B.1.617/B.1 or A strains, and all of them had similar genomic variations in spike proteins as well as non-structural proteins (NSPs). The mutations seen in these sequences in the Spike (S), NSPs, and open reading frame (ORF) regions would have produced amino acid changes known to improve viral replication, confer drug resistance, influence host-cell interaction, and lead to antigenic drift. CONCLUSIONS: Increased virulence culminating in vaccine immunity evasion may be inferred from these specific mutations. Our study adds to the growing body of evidence linking rapidly emerging mutations in the S (Spike) and ORF genes of the SARS-CoV-2 genome to immune evasion.

Novel 5,6-diphenyl-1,2,4-triazine-3-thiol derivatives as dual COX-2/5-LOX inhibitors devoid of cardiotoxicity.

A novel series of 5,6-diphenyl-1,2,4-triazine-3-thiol derivatives were designed, synthesized, and screened for their inhibitory potential against COX-2 and 5-LOX enzymes. The compounds from the series have shown moderate to excellent inhibitory potential against both targets. Compound 6k showed the inhibitions against COX-2 (IC50 = 0.33 ± 0.02 µM) and 5-LOX inhibition (IC50 = 4.90 ± 0.22 µM) which was better than the standard celecoxib (IC50 = 1.81 ± 0.13 µM) for COX-2 and zileuton (IC50 = 15.04 ± 0.18 µM) for 5-LOX respectively. Further investigation on the selected derivative 6k in rat paw edema models revealed significant anti-inflammatory efficacy. Compound 6k has also shown negligible ulcerogenic liability as compared to indomethacin. Moreover, in vivo biochemical analysis also established the compound's antioxidant properties. Compounds 6c and 6k were also observed to be devoid of cardiotoxicity post-myocardial infarction in rats. The molecular docking and dynamics simulation studies of the most active derivative 6k affirmed their consentient binding interactions with COX-2 specific ravine and cleft of 5-LOX.

COVID-19: Impact on linguistic and genetic isolates of India.

The rapid expansion of coronavirus SARS-CoV-2 has impacted various ethnic groups all over the world. The burden of infectious diseases including COVID-19 are generally reported to be higher for the Indigenous people. The historical knowledge have also suggested that the indigenous populations suffer more than the general populations in the pandemic. Recently, it has been reported that the indigenous groups of Brazil have been massively affected by COVID-19. Series of studies have shown that many of the indigenous communities reached at the verge of extinction due to this pandemic. Importantly, South Asia also has several indigenous and smaller communities, that are living in isolation. Till date, despite the two consecutive waves in India, there is no report on the impact of COVID-19 for indigenous tribes. Since smaller populations experiencing drift may have greater risk of such pandemic, we have analysed Runs of Homozygosity (ROH) among South Asian populations and identified several populations with longer homozygous segments. The longer runs of homozygosity at certain genomic regions may increases the susceptibility for COVID-19. Thus, we suggest extreme careful management of this pandemic among isolated populations of South Asia.

To Determine the Genotyping of Fc-gamma Receptor FCGR2A Polymorphism as Genetic Susceptibility to Neonatal Sepsis: A Study from a Tertiary Center of North India.

Background: Neonatal sepsis term is an infection of newborns <28 days of age. It is a common cause of death in developing countries. The receptor-gamma receptor FCGR2A has been shown to be associated with neonatal sepsis. It is an activating receptor found in many cell types such as monocytes, neutrophils, macrophages, platelets, and others. The receptor has a polymorphism (single-nucleotide polymorphism rs1801274) in its gene (FCGR2A) that encodes either a histidine (H) or arginine (R) at amino acid position 131. There are many studies showing the impact of these FCGR2A polymorphisms on sepsis. Our study aims to determine the prevalence of Fc-gamma receptor FCGR2A (rs1801274) polymorphism in neonatal sepsis and control in Eastern UP populations. Patients and Methods: We conducted a cross-sectional descriptive study of 590 patients (310 healthy individuals and 280 sepsis patients) to determine polymorphisms in the CD32A coding region in neonates. All individuals were genotyped for a variant at position 131 of the FcγRIIA gene. Discussion: In our study, the prevalence of FcγRIIa polymorphism is more in neonates with sepsis than in noninfected neonates. It was observed that the heterozygous allele (AG) were significantly increased in septic neonates when compared to the normal. Conclusion: Our data indicate that FcγRIIA genotyping can be used as a marker of genetic susceptibility to sepsis.

The major genetic risk factor for severe COVID-19 does not show any association among South Asian populations.

With the growing evidence on the variable human susceptibility against COVID-19, it is evident that some genetic loci modulate the severity of the infection. Recent studies have identified several loci associated with greater severity. More recently, a study has identified a 50 kb genomic segment introgressed from Neanderthal adding a risk for COVID-19, and this genomic segment is present among 16% and 50% people of European and South Asian descent, respectively. Our studies on ACE2 identified a haplotype present among 20% and 60% of European and South Asian populations, respectively, which appears to be responsible for the low case fatality rate among South Asian populations. This result was also consistent with the real-time infection rate and case fatality rate among various states of India. We readdressed this issue using both of the contrasting datasets and compared them with the real-time infection rates and case fatality rate in India. We found that the polymorphism present in the 50 kb introgressed genomic segment (rs10490770) did not show any significant correlation with the infection and case fatality rate in India.

Correlation of follicle-stimulating hormone receptor gene Asn 680 Ser (rs6166) polymorphism with female infertility.

BACKGROUND AND AIMS: Female infertility is a complex multifactorial, and polygenic disease associated with genetic factors plays an essential role in its formation and follicle development, oocyte maturation, and steroidogenesis regulation in the ovary. The aim here is too study the genetic association between follicle-stimulating hormone receptor (FSHR) Asn680Ser; (rs6166) gene polymorphism with female Infertility in our population. METHODS: In this prospective case-control study, we enrolled 106 infertile and 164 unrelated healthy control individuals. Genomic DNA was extracted from the 5 ml of venous blood using the modified salting-out method. A polymerase chain reaction-amplified exon 10 of FSHR and purified PCR products were sequenced on an ABI 3730XL DNA sequencer. The data were analyzed statistically. RESULTS: We found that the presence of rare allele "G" and heterozygous and common homozygous genotypes significantly increased the risk of female infertility. No significant change in the FSHR 191756 G >A genotype frequency was observed, regardless of chromosomal integrity. The genotype frequency distribution of locus 680 was consistent with the Hardy-Weinberg Equilibrium (HWE) in both groups (P > 0.05). CONCLUSION: No significant differences were found in allelic variants frequency and genotype distribution between each category of subjects when analyzing the FSHR SNPs in the exonic region (P value >0.05). FSHR Asn680Ser polymorphisms and female infertility (P > 0.05). Variations in FSHR gene have an essential influence on ovarian function and can account for several defects of female fertility. FSHR Asn680Ser (rs6166) gene polymorphism is associated with female infertility and can be used as a relevant molecular biomarker to identify the risk of infertility in our population. This finding can be important for disease pathogenesis.

Copy number variation of LINGO1 in familial dystonic tremor.

OBJECTIVE: To elucidate the genetic cause of a large 5 generation South Indian family with multiple individuals with predominantly an upper limb postural tremor and posturing in keeping with another form of tremor, namely, dystonic tremor. METHODS: Whole-genome single nucleotide polymorphism (SNP) microarray analysis was undertaken to look for copy number variants in the affected individuals. RESULTS: Whole-genome SNP microarray studies identified a tandem duplicated genomic segment of chromosome 15q24 present in all affected family members. Whole-genome sequencing demonstrated that it comprised a ∼550-kb tandem duplication encompassing the entire LINGO1 gene. CONCLUSIONS: The identification of a genomic duplication as the likely molecular cause of this condition, resulting in an additional LINGO1 gene copy in affected cases, adds further support for a causal role of this gene in tremor disorders and implicates increased expression levels of LINGO1 as a potential pathogenic mechanism.

Study of Dystrophinopathy in Eastern Uttar Pradesh Population of India.

INTRODUCTION: The frequency and distribution of dystrophin gene deletions vary in patients with Duchene/Becker muscular dystrophy (DMD/BMD). OBJECTIVE: In this study, we aimed to analyze clinical, biochemical, and dystrophin gene deletion pattern, by using multiplex polymerase chain reaction (PCR) in the population of eastern Uttar Pradesh and the adjoining districts of Bihar and Madhya Pradesh. MATERIAL AND METHOD: After clinical assessment, 225 patients of DMD/BMD were analyzed for deletion in dystrophin gene. Clinical features and biochemical parameters were noted. For genetic study, all samples were tested for deletion from 25 exons of DMD gene by using multiplex PCR. RESULT: Deletions were detected in 169 (75.1%) patients of DMD/BMD. Deletions were observed in both proximal and mid-distal hot spot regions with maximum deletion localized in the mid-distal hot spot region of the gene. The most frequent deletions were observed in exon 50 (14.9%) and exon 49 (10.8%). CONCLUSION: This study concludes that mid-distal region of dystrophin is highly polymorphic in the population of eastern Uttar Pradesh and responsible for pathogenesis of DMD. The population of eastern Uttar Pradesh shows similar pattern of deletion in dystrophin gene when compared with other ethnic groups of the Indian population.

Homocysteine and vitamin B12: Other causes of neural tube defects in Eastern Uttar Pradesh and Western Bihar population.

BACKGROUND: Neural tube defects (NTDs) are congenital malformations with an incidence of 1-10/1000 live births. Homocysteine and vitamin B12 metabolism have been shown to be associated with NTDs. AIM: To investigate the status of maternal and neonate's folic acid, homocysteine, and vitamin B12 levels and their association with the risk of development of NTDs in the population of Eastern Uttar Pradeshand Western Bihar, India. MATERIALS AND METHODS: This study is a cross-sectional, retrospective study where 96 mothers who either had a first NTD child or had a history of NTD child in the family and 126 neonates with spina bifida were recruited during the period 2012-2015. Eighty-four control mothers whose previous and current pregnancies were normal, and 87 control neonates who had no defects and were within the same age range as the NTD affected neonates, recruited from the department of pediatric surgery, were enrolled in the study. Plasma concentrations of folic acid, vitamin B12, and homocysteine were compared between cases and controls. RESULTS: The folic acid level in the mothers and neonates was within the normal limit. A significant increase in the level of homocysteine in mothers with affected pregnancy and in neonate cases in comparison to control mothers was obseved. Further, a significant decrease in the level of vitamin B12 in mothers with NTD neonates and in the affected neonates was noted. A negative correlation was found between homocysteine and vitamin B12 levels in case and control mothers. CONCLUSION: A correlation of an increase in serum homocysteine with a decrease in vitamin B12 was seen in mothers of neonates with NTD. A similar observation as made in the neonates with NTDs. It may be suggested that maternal decrease in vitamin B12, in mothers who have normal folic acid may be associated with NTD in their children.

A prospective pilot study on serum cleaved tau protein as a neurological marker in severe traumatic brain injury.

OBJECTIVE: Neurotrauma has been labelled as a "silent epidemic" affecting both the developed and the developing nations. To date, no single brain-specific biomarker has been unanimously accepted for routine clinical use in TBI. Our study aims to determine the correlation of "cleaved-tau protein" in severe traumatic brain injury (TBI) with Glasgow Coma Scale (GCS) at the time of admission, mode of injury, CT findings and outcome at discharge. METHODS: The study has been approved by the institutional ethical committee. 40 cases with severe TBI and 40 randomly selected healthy controls were included in this prospective study. Venous blood samples were collected and serum cleaved tau protein levels were measured and correlated with gender, mode of injury, CT findings GCS score and GOS score at discharge. RESULTS: In the severe TBI group, the mean serum cleaved tau protein levels in males were 91.65 ± 41.34 pg/ml (mean ± S.D.), and females were 104.43 ± 53.08 pg/ml (mean ± S.D.), (p = 0.27). Mean serum C-tau level in study group was 95.48 ± 44.87 pg/ml (range 36.44-192.34), 95% C.I. (81.13-109.83) and in controls was 33.82 ± 13.65 pg/ml (range 2.48-66.54), 95% C.I. (29.46-38.19) (p < 0.001). The distribution of serum C-tau was in severe TBI group varied in all categories of GCS at 0th day (p < 0.001). Serum cleaved tau protein levels in the good outcome group were 74.26 ± 25.43 pg/ml (mean ± S.D.), range 36.44-144.54, 95% C.I. (63.52-85.00) and the poor-outcome group were 127.32 ± 49.40 pg/ml, range 66.65-192.34, 95% C.I. (100.99-153.64) (p = 0.001). CONCLUSION: In severe TBI, serum cleaved tau protein levels were significantly higher as compared to the controls in this prospective study. However, results of this study are preliminary in nature and there is a need to undertake larger prospective studies to reach a definitive conclusion.

PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment.

PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.

Effect of Tocopheryl Acetate on Maternal Cigarette Smoke Exposed Swiss Albino Mice Inbred Fetus.

INTRODUCTION: Cigarette smoking is worldwide problem which can be correlated with teratogenicity. Tocopheryl acetate plays as an antioxidant against the oxidative stress evolved by cigarette smoke exposure during pregnancy. AIM: To study the effect of maternal exposure to cigarette smoke and Tocopheryl acetate on fetuses of mice. MATERIALS AND METHODS: Pregnant mice randomly assigned to different groups (Group I (control), Group II (Tocopheryl acetate), Group III(soyabean oil used as vehicle for Tocopheryl acetate), Group IV (Cigarette smoke Exposed), Group V (Cigarette smoke exposed plus Tocopheryl acetate) and Group VI(Cigarette smoke exposed plus soyabean oil) were exposed to cigarette smoke 3 times a day for 20 minutes each time and Tocopheryl acetate with dose of 200mg/kg/day in 0.3ml of soyabean oil as vehicle orally through oral gavage from the 5th day of gestation to 15th day. RESULTS: Cigarette smoke exposed mice showed significant fetal weight loss, resorption, placental anomalies, severe growth retardation, venous congestion, haemorrhage, limbs defects and enphalocele. Negligible abnormalities were seen among the control and Tocopheryl acetate group. Cigarette smoke exposed group with Tocopheryl acetate exhibited weight gain among the fetus as well as no gross abnormalities. The oxidative stress was significantly increased by increasing Malondialdehyde (MDA) 293±81.57 µmol/mg (p<0.0001) and decreasing Superoxide Dismutase (SOD) 1.43 ± 0.23mg/ml, (p<0.0001) Reduced Glutathione (GR) 0.017±0.002mg/ml, (p<0.01) and Catalase (CAT) 0.248±0.005mg/ml, (p<0.0001). Tocopheryl acetate induced group significantly maintained the oxidative stress with all p <0.0001. CONCLUSION: It can be concluded that Tocopheryl acetate may have an ameliorating effect on the cigarette smoke during pregnancy on fetus.