Transoral diode laser-assisted resection of a tongue base schwannoma.

Schwannomas are benign peripheral nerve sheath tumours with a low risk of malignant transformation. About 25%-40% are in the head and neck region with the posterior third of the tongue being a rather rare site of its growth, and a lesion at this site is understandably difficult to approach and treat. Being benign and encapsulated, surgical excision is in the mainstay of treatment. Symptoms can range from a globus pharyngeus and dysphagia to airway compromise depending on the size and site. Traditionally, a paramedian lip split approach with paramedian mandibulotomy and mandible swing may be used. However, some recent reports of the use of carbon dioxide laser and robotic surgery for tongue base lesions are seen in the literature. Our case report is a unique addition to the management strategies for such tongue base lesions as we employed diode 980 nm laser using a minimally invasive transoral approach with a successful outcome.

The tobacco chloroplast YCF4 gene is essential for transcriptional gene regulation and plants photoautotrophic growth.

A tobacco chloroplast hypothetical open reading frame 4 (YCF4) has been reported as a non-essential assembly factor for photosynthesis based on an incomplete knockout of YCF4, just 93 of 184 amino acids from the N-terminus were knocked out. On the other hand, we removed the complete sequence of YCF4 from tobacco chloroplasts and observed that ΔYCF4 plants were unable to survive photoautotrophically as their growth was hampered in the absence of an external carbon supply, clearly showing that the YCF4 is essential for photosynthesis. Initially, the aadA gene was introduced into the tobacco plastome replacing the complete YCF4 gene through homologous recombination events. The replacement of YCF4 with aadA was confirmed by PCR and Southern blot analysis in ΔYCF4 plants. Homoplasmic ΔYCF4 plants had a light green phenotype, and the leaves became pale yellow as the plants grew older. The structure of chloroplasts of ΔYCF4 mutants of light green phenotype was studied using a transmission electron microscope (TEM), and the micrographs demonstrated structural anomalies in the chloroplasts; including shape, size, and grana stacking compared to the wild-type plants. Further, transcriptome analysis revealed that the expression of PSI, PSII, and ribosomal genes remained unchanged in ∆YCF4 plants. On the other hand, transcriptome levels of rbcL (Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit), LHC (Light-Harvesting Complex), and ATP Synthase (atpB and atpL) decreased, indicating that the YCF4 has the function(s) in addition to assembling the photosynthetic complex. This was confirmed by in-silico protein-protein interactions of full-length YCF4 as well as 93 and 91 of 184 amino acids from N- and C-termini of the full-length protein, which revealed that the C-terminus (91 aa) of YCF4 is important in interacting with other chloroplast proteins. These findings provide genetic support for the plastid YCF4 gene's critical role in regulating the plastid gene expression and assembling the photosynthetic complex.

Endonasal endoscopic laser-assisted resection of septal glomangiopericytoma.

Glomangiopericytoma is a rare vascular neoplasm characterised by a pattern of prominent perivascular growth with myoid phenotype. It is categorised as a borderline low-malignancy tumour by WHO and accounts for less than 0.5% of all sinonasal tumours. After curative resection, patients of glomangiopericytoma need long-term endoscopic follow-up due to high risk of recurrence.We report a case of a 23-year-old man complaining of nasal obstruction off and on and frequent epistaxis. A reddish mass in the right nasal cavity was observed on endoscopy and treated with endoscopic excision.Biopsy revealed this to be glomangiopericytoma arising from the septum of right nasal cavity, which was excised in toto with endonasal endoscopic approach using diode laser.

Metabolic engineering of the chloroplast genome reveals that the yeast ArDH gene confers enhanced tolerance to salinity and drought in plants.

Osmoprotectants stabilize proteins and membranes against the denaturing effect of high concentrations of salts and other harmful solutes. In yeast, arabitol dehydrogenase (ArDH) reduces D-ribulose to D-arabitol where D-ribulose is derived by dephosphorylating D-ribulose-5-PO4 in the oxidized pentose pathway. Osmotolerance in plants could be developed through metabolic engineering of chloroplast genome by introducing genes encoding polyols since chloroplasts offer high level transgene expression and containment. Here, we report that ArDH expression in tobacco chloroplasts confers tolerance to NaCl (up to 400 mM). Transgenic plants compared to wild type (WT) survived for only 4-5 weeks on 400 mM NaCl whereas plants remained green and grew normal on concentrations up to 350 mM NaCl. Further, a-week-old seedlings were also challenged with poly ethylene glycol (PEG, up to 6%) in the liquid medium, considering that membranes and proteins are protected under stress conditions due to accumulation of arabitol in chloroplasts. Seedlings were tolerant to 6% PEG, suggesting that ARDH enzyme maintains integrity of membranes in chloroplasts under drought conditions via metabolic engineering. Hence, the gene could be expressed in agronomic plants to withstand abiotic stresses.

Entamoeba infections in different populations of dogs in an endemic area of Lahore, Pakistan.

Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI=±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI=±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P≤0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI=±7.86) and 11 (5.6%, 95% CI=±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI=±6.58) and 23 (18.8%, 95% CI=±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI=±5.62) and 32 (11.3%, 95% CI=±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P≤0.05) between all age groups. Statistically, no significant (P≥0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI=±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI=±10.4) and crossbred (19 of 239, 8.3%, 95% CI=±7.47), indicating a significant (P≤0.05) trend of positivity between various breeds of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen.

Prevalence of Entamoeba histolytica -like cysts compared to E. histolytica antigens detected by ELISA in the stools of 600 patients from three socioeconomic communities in the Metropolitan City of Lahore, Pakistan.

Amoebiasis, caused by Entamoeba histolytica , has a worldwide distribution and is of public health significance in many developing countries. It has a fecal-oral transmission cycle and is most prevalent in developing countries in regions where substandard sanitary conditions exist due to poverty. Little is known about the epidemiology of E. histolytica infection and its presence in different socioeconomic communities in developing countries. We undertook the present study in the city of Lahore, Pakistan, and our prediction was that the prevalence of E. histolytica -like cysts and E. histolytica stool antigen would be lower in patients from upper socioeconomic levels than in individuals from middle or lower socioeconomic levels. We investigated the prevalence of E. histolytica in humans from 3 socioeconomic communities in territories of Lahore, Pakistan. Six hundred fecal samples were collected and examined using both microscopy (triple fecal test) to detect cysts of E. histolytica -like amoeba and ELISA (stool antigen ELISA) to demonstrate diagnostic stool antigens of E. histolytica . Samples were from individuals living under conditions deemed to be upper socioeconomic class (n = 287), middle socioeconomic class (n = 172), and lower socioeconomic class (n = 141). The total prevalence of positive samples was 22.5% (135/600) by triple test and 16.8% (101/600) by stool antigen ELISA in the 600 fecal samples. Statistically, significant (P < 0.05) differences in prevalence were seen between the 3 socioeconomic class groups. Forty-four (15.3%) and 32 (11.1%) of 287 in the fecal samples from the upper socioeconomic class were positive by triple test and by antigen ELISA, respectively. Thirty-nine (22.6%) and 29 (16.8%) of 172 in the fecal samples from the middle socioeconomic class were positive by the triple test and by antigen ELISA, respectively. Fifty-two (36.8%) and 40 (28.3%) of 141 in the fecal samples from the lower socioeconomic class were positive by the triple test and by antigen ELISA, respectively. We accept our hypothesis based on these findings. We also demonstrated that fecal samples collected from the youngest age group (1 mo-5 yr) were more likely to be positive for E. histolytica antigens than were samples from the other 3 age groups, and that prevalence was significantly higher (P < 0.05) in the summer than in the other 3 seasons. These results highlight the importance of surveillance of this relatively ignored pathogen in this developing metropolitan city in Pakistan.

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum.

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.

Synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant.

The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. In the present study the interferon alpha 5 gene was synthesized by using complex set of oligos. After sequence confirmation of the synthesized gene, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 gene. The recombinant fragment was then tethered with chloroplast light inducible promoter, rbcl followed by sequential cloning to develop chloroplast transformation vector to target the cassette into the inverted repeat region of plastome through two events of homologous recombination. The putative transgenic plants obtained through biolistic delivery method and as a result of antibiotic selection of bombarded leaves, were subjected to different rounds of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were analyzed through Polymerase Chain Reaction and Sothern blotting. The expression of introduced synthetic genes was recorded using Enzyme Linked ImmunoSorbant Assay technique. It was experienced that mature leaves contained comparatively high levels of interferon compared to young and senescence leaves.

Relationship of age to body weight, scrotal circumference, testicular ultrasonograms, and semen quality in Sahiwal bulls.

The objective of the present study was to determine the relationship of age to body weight (BW), scrotal circumference (SC), number of pixels of testicular ultrasonograms (NP), and semen quality in Sahiwal bulls. The study was based on 128 Sahiwal bulls of different age groups (from 0 to >100 months of age). Bulls were evaluated for SC, BW, and NP. Semen was evaluated once a week for five consecutive weeks from regularly collected donor bulls (n = 86) ranging in age from 25-30 to >100 months. Ejaculate volume, sperm motility, sperm concentration, sperm morphology, percent live sperms, sperm plasma membrane integrity, and normal acrosome were compared among different age groups. Mean SC and BW increased (P < 0.05) in a curvilinear manner from birth to >100 months of age. Mean NP of testicular ultrasonograms increased (P < 0.05) from 0 to 24 months and then plateaued until >100 months of age. Body weight, SC, and NP were positively correlated with age from birth until >100 months (r = 0.91, 0.87, and 0.40, respectively). Ejaculate volume (5.7 ± 0.2 vs. 4.6 ± 0.09 ml) and sperm concentration (1,281.6 ± 17.7 vs. 1,115.8 ± 55.9 × 10(6)/ml) increased (P < 0.05) in mature bulls compared to younger ones. However, motility (68.6 ± 0.3%), plasma membrane integrity (50.8 ± 1.0%), and normal acrosome (74.8 ± 0.5%) remained insignificant due to age. In six of eight age groups studied, morphological abnormalities were well within the range (18.1 ± 0.3%). In conclusion, the BW, SC, and NP of testicular ultrasonograms, ejaculate volume, and concentration increased with age. Moreover, semen quality is fairly independent of age except volume and concentration in Sahiwal bulls.

Codon optimization of cry1Ab gene for hyper expression in plant organelles.

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.

Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

Metabolic engineering of the chloroplast genome using the Echerichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis.

p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5' untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.