Telomere length in dromedary camels (Camelus dromedarius) produced by somatic cell nuclear transfer (SCNT) and their age-matched naturally produced counterparts.

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Infantile thiamine deficiency: Redefining the clinical patterns.

OBJECTIVES: Thiamine deficiency (TD) is frequently suspected and treated at our hospital. In our retrospective study, we aimed at finding the clinical and laboratory spectrum of infantile TD presenting to a single center over a period of time. METHODS: The diagnosis was made on criterion standard of response to thiamine challenge. RESULTS: TD was suspected in 189 infants at admission; 43 infants were diagnosed as having TD in three distinct forms and a fourth group with mixed presentation. The first group (n = 30), which was the youngest (mean age = 67 d), was always associated with lactic acidosis. They had history of reflux and suddenly became irritable and developed acidotic breathing. This further worsened into shock (46%) and acute respiratory failure (50%). The second group (n = 5) presented with pulmonary arterial hypertension. They had hoarseness of voice and irritability. Chest radiograph showed prominent pulmonary conus. Their clinical course was complicated by congestive heart failure in three. Echocardiographic response to thiamine was uniformly seen within 3 d in this group. The clinical presentation of infants with Wernicke's encephalopathy (n = 5) who were the oldest of all (mean age = 190 d) was constantly marked by presence of bilateral ptosis and encephalopathy preceded by occurrence of vomiting. Their head ultrasonography showed presence of hyperechoic basal ganglia. CONCLUSIONS: Three clinically distinct forms of TD were recognized. Lactic acidosis was a universal finding in acidotic form. Infants with pulmonary hypertension as primary presentation are typically associated with aphonia. Infants with Wernicke's encephalopathy can be clinically diagnosed by presence of encephalopathy and ophthalmic signs (ptosis).

In vitro embryo production (IVEP) in camelids: Present status and future perspectives.

Camels are a fundamental livestock resource with a significant role in the agricultural economy of dry regions of Asia and Africa. Similarly, llamas and alpacas are an indigenous resource considered as beasts of burden in South America because of their surefootedness and ability to adapt. Camel racing, a highly lucrative and well-organized sport, camel beauty contests, and high demand for camel milk lead to a steady interest in the multiplication of elite animals by in vitro embryo production (IVEP) in this species during the last few decades. Although offspring have been produced from in vitro produced embryos, the technique is still not that well developed compared with other domestic animal species such as cattle. IVEP involves many steps, including the collection of oocytes from either slaughterhouse ovaries or live animals through ultrasound-guided transvaginal aspiration; in vitro maturation of these collected oocytes; collection and preparation of semen for fertilization; culture and passaging of cells for nuclear transfer, chemical activation of the reconstructed embryos, and in vitro culture of embryos up to the blastocyst stage for transfer into synchronized recipients to carry them to term. This review discusses the present status of all these steps involved in the IVEP of camelids and their future perspectives.

Development of prepubertal goat oocytes after their in vitro maturation and chemical activation.

Experiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus-oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

Effect of roscovitine pretreatment on in vitro maturation of oocytes and their subsequent developmental after chemical activation in dromedary camel (Camelus dromedarius).

Studies were conducted to evaluate an optimal concentration of roscovitine needed to maintain abattoir origin oocytes at germinal vesicle stage in experiment 1 and their subsequent maturation and developmental competence after chemical activation in experiments 2 and 3, respectively. The cumulus-oocyte complexes (COCs) aspirated from ovaries collected from a local slaughterhouse were cultured in TCM-199 based pre-maturation medium supplemented with 25, 50 or 75 µM roscovitine, depending on the experimental group. After 24 h, the COCs were denuded of cumulus, fixed and stained with aceto-orcein and examined for their nuclear status. They were classified as germinal vesicle, diakinesis, metaphase-I, metaphase-II and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. In experiment 2, the COCs pre-matured in media supplemented with 50 µM roscovitine for 24 h were washed and kept for in vitro maturation along with another group of freshly collected COCs for 30 h. All the oocytes were fixed and stained to evaluate their nuclear status as described above. In experiment 3, all mature oocytes obtained from the COCs pre-matured in media supplemented with 50 µM roscovitine and those obtained from freshly collected group were activated by 5 mM ionomycin. Activated oocytes were cultured in embryo culture medium for a period of 7 days to evaluate their developmental potential. The proportion of oocytes at GV stage in the group pre-matured in media with 50 µM-was significantly (P < 0.01) higher when compared with the group having 25 µM of roscovitine. No difference was found in the proportion of GV stage oocytes in this group when compared with the freshly collected COCs. None of the oocytes reached to M-II stage in any of the three treatment groups. In experiment 2, no difference was observed in the proportion of oocytes reaching M-II stage between the groups after 30 h of in vitro culture; however, higher proportion of oocytes (P < 0.05) were classified as others in the pre-maturation group when compared with the group having freshly collected oocytes. In experiment 3, no difference was observed in the proportion of oocytes cleaving and those developing to the blastocyst stage between the pre-matured and freshly matured groups. In conclusion, the present study, for the first time, demonstrates the possible use of roscovitine as a meiotic inhibitor for camel oocytes. Keeping in view the ability of these oocytes to mature and develop to the blastocyst stage at par with the fresh oocytes, more flexible schedules for maturation and manipulation of such oocytes could be developed.

First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels.

Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5µM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term.

Thiamine responsive acute life threatening metabolic acidosis in exclusively breast-fed infants.

BACKGROUND: Acute life threatening metabolic acidosis in exclusively breast fed infants due to thiamine deficiency is not described. Kashmir valley, a north Indian state has a population that largely consumes polished rice. METHODS: A six months prospective descriptive study of infants who presented with acute life threatening metabolic acidosis (Blood pH ≤ 7.0) due to thiamine deficiency. RESULTS: Twenty three infants (Eleven male; Twelve female) in the age range of 32 days to 4 months had a pH of ≤7 at admission. Onset of moaning was immediate (2-24 hours). Blood lactate levels were more than 15mmol/L. Blood thiamine levels of six infants in whom it was done ranged from 11-69 nmol/L (control 78-185 nmol/L). All infants were exclusively breast fed. Maternal staple diet consisted of polished rice. All mothers consumed rice after washing it thrice. Twelve lactating mothers were on customary dietary restrictions. Practice of straining rice after cooking was observed in thirteen. The commonest symptoms were irritability (82%) and reflux (56%). Commonest signs were tachycardia (100%) and moaning (73%). At presentation 52% were in cardiogenic shock. Response to thiamine was dramatic with moaning and irritability subsiding in two hours and tachycardia in four hours. Adequate perfusion was achieved in one hour. Eighteen patients seen at six months follow up had normal neurodevelopment. CONCLUSIONS: Thiamine deficiency in an infant can present as sudden onset metabolic acidosis. If treated early, metabolic acidosis due to thiamine deficiency is associated with good immediate and long term prognosis even if pH is less than 7 at presentation.

Recurrent meningitis in an adult secondary to an inner ear malformation: imaging demonstration.

Congenital labyrinthine dysplasia with a translabyrinthine cerebrospinal fluid (CSF) fistula may be an anatomic cause for recurrent meningitis. This condition is usually seen in children aged 5 to 10 years who present with sensorineural hearing loss (SNHL) and CSF discharge through the nose or ear, with or without recurrent meningitis. Multidetector-row computed tomography (MDCT) and high-resolution T2-weighted magnetic resonance imaging (MRI) of the petrous portion of the temporal bone can help to diagnose this abnormality. We report a case of translabyrinthine CSF fistula in an adult-a 30-year-old man-who presented with recurrent pneumococcal meningitis, a long history of a clear nasal discharge, and evidence of SNHL. MDCT and MRI of the temporal bone demonstrated a cystic-appearing cochleovestibular malformation (an incomplete partition type I) in the right inner ear. Imaging also showed an absence of the basal turn of the cochlea and the cribriform membrane at the lateral end of the right internal auditory canal, which was shorter and narrower than normal. Evidence of fluid in the right middle ear suggested a CSF fistula.

Subcutaneous emphysema due to bronchial foreign body demonstrated by multidetector-row computed tomography.

Foreign body aspiration is an important cause of emergency hospital admissions in young children less than 3 years of age. It may manifest with acute respiratory difficulty, choking and wheeze acutely or may be asymptomatic. Surgical emphysema is an unusual presentation of bronchial foreign body aspiration in young children. We describe an infant with bronchial foreign body aspiration that manifested with subcutaneous emphysema and pneumomediastinum. Multidetector-row CT with virtual bronchoscopy helped in the diagnosis by detecting and localizing the intraluminal foreign body in the right main bronchus that was removed with rigid bronchoscopy.

Malignant solid pseudopapillary tumor of pancreas causing sinistral portal hypertension.

Solid pseudopapillary tumor (SPT) of the pancreas is a rare benign or low-grade malignant epithelial tumor that occurs mainly in young females in second to fourth decades of life. Pathologic and imaging findings include a well-defined, encapsulated pancreatic mass with cystic and solid components with evidence of hemorrhage. We report a 23-year-old female who presented with upper abdominal pain of long duration and epigastric mass on palpation. Multidetector-row CT (MDCT) demonstrated a large well-defined heterogeneous attenuation mass, containing hyperdense areas of hemorrhage mixed with solid enhancing and cystic non-enhancing areas, arising from the pancreatic body and tail. Splenic vein thrombosis was present with dilated splenoportal collateral vessels between splenic hilum and portal/superior mesenteric veins, with dilated vessels seen in the gastric wall, with patent portal vein, compatible with sinistral portal hypertension. Typical imaging features and age and sex of the patient suggested a diagnosis of SPT of pancreas complicated by segmental portal hypertension due to splenic vein thrombosis. Histopathology of the biopsy material was confirmatory.

Klebsiella brain abscess in a neonate.

Klebsiella pneumoniae is rarely associated with neonatal cerebral abscess. A case of Klebsiella brain abscess in a neonate is described. Diagnosis of abscess was confirmed by magnetic resonance imaging and ultrasound-guided aspiration.

Giant adrenal myelolipoma: Incidentaloma with a rare incidental association.

Adrenal myelolipoma is an unusual, benign and biochemically inactive tumor that is composed of mature adipose and hematopoietic tissue. It is usually diagnosed accidentally and nowadays much more frequently because of widespread use of ultrasonography, computed tomography (CT) and magnetic resonance imaging. Adrenal myelolipoma is usually unilateral and asymptomatic, though known to be associated with obesity, hypertension, endocrinological disorders and some malignancies. We report herein two cases of right-sided giant adrenal myelolipoma diagnosed by multidetector-row CT. One patient was symptomatic because of a large mass in the right upper abdomen, which on imaging with CT was seen to be right adrenal myelolipoma. Another patient had a large left side Bochdalek hernia and right adrenal myelolipoma was incidentally discovered on CT.

Foreign body within sphenoid sinus: multidetector-row computed tomography (MDCT) demonstration.

Trans-nasal sphenoid sinus foreign body is a rare condition. We report a case of trans-nasal gun bullet within the sphenoid sinus with breach of the floor of the sella turcica. A 42-year-old soldier presented a few weeks after a gun battle with a history of headache. The skull x-ray demonstrated a foreign body in the region of sphenoid sinus and sella. Multidetector-row CT with multiplanar reformats demonstrated a bullet within the sphenoid sinus with its tip penetrating into the sella turcica. A few days later the patient coughed up the bullet as it came out spontaneously without any intervention. This is perhaps the unique occurrence of such a foreign body.

Perinephric abscess caused by ruptured retrocecal appendix: MDCT demonstration.

Acute appendicitis may occasionally become extraordinarily complicated and life threatening yet difficult to diagnose. One such presentation is described in a 60-year-old man who was brought to the hospital due to right lumbar pain and fever for the last 15 days. Ultrasonography showed a right perinephric gas and fluid collection. Abdominal computed tomography with multidetector-row CT (MDCT) revealed gas-containing abscess in the right retroperitoneal region involving the perinephric space, extending from the lower pole of the right kidney up to the bare area of the liver. Inflamed retrocecal appendix was seen on thick multiplanar reformat images with its tip at the lower extent of the abscess. Laparotomy and retroperitoneal exploration were performed immediately and a large volume of foul smelling pus was drained. A ruptured retrocecal appendix was confirmed as the cause of the abscess.

Parry-Romberg syndrome associated with unusual intracranial vascular malformations and Phthisis bulbi.

Neurovascular anomalies of Parry-Romberg syndrome have been reported infrequently. We report a case of Parry-Romberg syndrome with hypoplastic left internal carotid, middle cerebral, anterior cerebral, posterior communicating and posterior cerebral artery. The patient presented with partial seizures, hemiparesis and phthisis bulbi.

A study on p53 gene alterations in esophageal squamous cell carcinoma and their correlation to common dietary risk factors among population of the Kashmir valley.

AIM: To systematically examine the extent of correlation of risk factors, such as age, consumed dietary habit and familial predisposition with somatic Tp53 molecular lesion causal to elevate carcinogenesis severity of esophageal squamous cell carcinoma (ESCC) among the Kashmiri population of Northern India. METHODS: All cases (n = 51) and controls (n = 150) were permanent residents of the Kashmir valley. Genetic alterations were determined in exons 5-8 of Tp53 tumor suppressor gene among 45 ESCC cases histologically confirmed by PCR-SSCP analysis. Data for individual cancer cases (n = 45) and inpatient controls (n = 150) with non-cancer disease included information on family history of cancer, thirty prevailing common dietary risk factors along with patient's age group. Correlation of genetic lesion in p53 exons to animistic data from these parameters was generated by Chi-square test to all 45 histologically confirmed ESCC cases along with healthy controls. RESULTS: Thirty-five of 45 (77.8%) histologically characterized tumor samples had analogous somatic mutation as opposed to 1 of 45 normal sample obtained from adjacent region from the same patient showed germline mutation. The SSCP analysis demonstrated that most common p53 gene alterations were found in exon 6 (77.7%), that did not correlate with the age of the individual and clinicopathological parameters but showed significant concordance (P<0.05) with familial history of cancer (CD = 58), suggesting germline predisposition at an unknown locus, and dietary habit of consuming locally grown Brassica vegetable "Hakh" (CD = 19.5), red chillies (CD = 20.2), hot salty soda tea (CD = 2.37) and local baked bread (CD = 1.1). CONCLUSION: Our study suggests that somatic chromosomal mutations, especially in exon 6 of Tp53 gene, among esophageal cancer patients of an ethnically homogenous population of Kashmir valley are closely related to continued exposure to various common dietary risk factors, especially hot salty tea, meat, baked bread and "Hakh", that are rich in nitrosoamines and familial cancer history.