Covalent Organic Frameworks for the Purification of Recombinant Enzymes and Heterogeneous Biocatalysis.

Recombinant enzymes have gained prominence due to their diverse functionalities and specificity and are often a greener alternative in biocatalysis. This context makes purifying recombinant enzymes from host cells and other impurities crucial. The primary goal is to isolate the pure enzyme of interest and ensure its stability under ambient conditions. Covalent organic frameworks (COFs), renowned for their well-ordered structure and permeability, offer a promising approach for purifying histidine-tagged (His-tagged) enzymes. Furthermore, immobilizing enzymes within COFs represents a growing field in heterogeneous biocatalysis. In this study, we have developed a flow-based technology utilizing a nickel-infused covalent organic framework (Ni-TpBpy COF) to combine two distinct processes: the purification of His-tagged enzymes and the immobilization of enzymes simultaneously. Our work primarily focuses on the purification of three His-tagged enzymes ß-glucosidase, cellobiohydrolase, and endoglucanase as well as two proteins with varying molecular weights, namely, green fluorescent protein (27 kDa) and BG Rho (88 kDa). We employed Ni-TpBpy as a column matrix to showcase the versatility of our system. Additionally, we successfully obtained a Ni-TpBpy COF immobilized with enzymes, which can serve as a heterogeneous catalyst for the hydrolysis of p-nitrophenyl-ß-d-glucopyranoside and carboxymethylcellulose. These immobilized enzymes demonstrated catalytic activity comparable to that of their free counterparts, with the added advantages of recyclability and enhanced stability under ambient conditions for an extended period, ranging from 60 to 90 days. This contrasts with the free enzymes, which do not maintain their activity as effectively over time.

Hierarchical covalent organic framework-foam for multi-enzyme tandem catalysis.

Covalent organic frameworks (COFs) are ideal host matrices for biomolecule immobilization and biocatalysis due to their high porosity, various functionalities, and structural robustness. However, the porosity of COFs is limited to the micropore dimension, which restricts the immobilization of enzymes with large volumes and obstructs substrate flow during enzyme catalysis. A hierarchical 3D nanostructure possessing micro-, meso-, and macroporosity could be a beneficial host matrix for such enzyme catalysis. In this study, we employed an in situ CO2 gas effervescence technique to induce disordered macropores in the ordered 2D COF nanostructure, synthesizing hierarchical TpAzo COF-foam. The resulting TpAzo foam matrix facilitates the immobilization of multiple enzymes with higher immobilization efficiency (approximately 1.5 to 4-fold) than the COF. The immobilized cellulolytic enzymes, namely ß-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (EG), remain active inside the TpAzo foam. The immobilized BGL exhibited activity in organic solvents and stability at room temperature (25 °C). The enzyme-immobilized TpAzo foam exhibited significant activity towards the hydrolysis of p-nitrophenyl-ß-d-glucopyranoside (BGL@TpAzo-foam: Km and Vmax = 23.5 ± 3.5 mM and 497.7 ± 28.0 µM min-1) and carboxymethylcellulose (CBH@TpAzo-foam: Km and Vmax = 18.3 ± 4.0 mg mL-1 and 85.2 ± 9.6 µM min-1 and EG@TpAzo-foam: Km and Vmax = 13.2 ± 2.0 mg mL-1 and 102.2 ± 7.1 µM min-1). Subsequently, the multi-enzyme immobilized TpAzo foams were utilized to perform a one-pot tandem conversion from carboxymethylcellulose (CMC) to glucose with high recyclability (10 cycles). This work opens up the possibility of synthesizing enzymes immobilized in TpAzo foam for tandem catalysis.

Neurogenic vision loss: Causes and outcome. An experience from a tertiary center in Northern India.

INTRODUCTION: Vision loss can be a consequence of numerous disorders of eye and neural pathway conveying visual input to brain. A variety of conditions can affect visual pathway producing neurogenic vision loss. The presentation and course of vision loss depends on the site of involvement and underlying etiology. We conducted this unprecedented study to evaluate the characteristics and outcome of various diseases of the visual pathway. MATERIALS AND METHODS: In this prospective cohort study, we evaluated 64 patients with neurogenic visual impairment. Ophthalmological causes were excluded in all of them. Their presentation, ophthalmological characteristics and investigation findings were recorded. These patients were followed up till 6 months. RESULTS: Out of 69 patients evaluated, 5 were excluded as they had ophthalmological abnormalities. The remaining 64 cases (113 eyes) were enrolled. 54 cases were due to diseases of anterior visual pathway and rest 10 had cortical vision loss. The etiologic distribution is as follows: Isolated optic neuritis- 12 (19%), multiple sclerosis- 4 (6.3%), neuromyelitis optica- 5 (7.9%), tubercular meningitis- 15 (23.8%), non-arteritic ischemic optic neuropathy, ischemic optic neuropathy complicating cavernous sinus thrombosis, cryptococcal meningitis, malignant infiltration of optic nerve, Crouzon's syndrome, calvarial thickening and traumatic occipital gliosis- 1 (1.6%) case each, idiopathic intracranial hypertension, pituitary adenoma, acute disseminated encephalomyelitis, posterior reversible leukoencephalopathy- 3 (4.8%) cases each, cortical venous thrombosis 5 (7.9%), subacute scleroing panencephalitis- 4 (6.3%) cases. CONCLUSIONS: The diseases of anterior visual pathway were much more common than cortical vision loss. A majority of our patients had severe impairment of vision at presentation.

Perihematomal edema as predictor of outcome in spontaneous intracerebral hemorrhage.

BACKGROUND: Spontaneous intracerebral hemorrhage (SICH) is a form of cerebrovascular accident with a very high rate of morbidity and mortality. The determinants of prognosis include the demographic, clinical, laboratory, and radiologic factors. It is long known that the hematoma size has a negative impact on the outcome in SICH. The influence of perihematomal edema (PHE) is not established to the extent same as that of hematoma volume. Hence, we planned this study to determine as to what role does PHE plays in the outcome in SICH. AIM OF THE STUDY: To evaluate the prognostic influence of absolute and relative edema (ratio of absolute edema to hematoma volume) in the patients of SICH. MATERIALS AND METHODS: This is a prospective case-controlled study. A total of 44 patients were enrolled after excluding the confounding factors. The patients were evaluated and their disability was assessed using modified Rankin scale (MRS). The imaging was done in the interval between 24 and 72 h and the hematoma volume, absolute edema volume, and the relative edema were calculated. The outcome was reassessed at 12 weeks and defined as favorable if MRS < 3. RESULTS: A total of 69 patients were found to be having SICH on imaging; however, 25 patients were excluded as they had one of the criterion for exclusion. Hence, only 44 patients were included in the study. On univariate analysis, none of the demographic characteristics of the patients, vascular risk factors, presenting complaints, blood pressure, Glasgow coma scale, and MRS at admission, laboratory parameters were not significantly different in the two outcome groups. The hematoma volume was significantly higher in the poor outcome group (P < 0.0001) and the relative edema was associated with a favorable outcome at 12 weeks (P < 0.0001). On multivariate logistic regression, the hematoma volume and relative edema were found to have effect on the outcome similar to that seen on univariate analysis. CONCLUSION: In SICH, a larger hematoma volume is a predictor of poor outcome and a relative edema is associated with a better functional status.

A case of extensive left-sided facial atrophy of Romberg.

Progressive facial atrophy or Parry-Romberg syndrome is characterized by slowly progressive facial atrophy involving skin, subcutaneous tissue, cartilage and bony structures. Apart from facial atrophy, it can be associated with diverse clinical manifestations including headache, partial seizures, trigeminal neuralgia, cerebral hemiatrophy and ocular abnormalities. The exact etiology is unknown although sympathetic system dysfunction, autoimmune disorders, focal scleroderma, trauma and genetic factors have been postulated. We hereby report a patient having marked left-sided facial atrophy and wasting of the tongue. Such an extensive wasting is not previously reported in the literature.

Thyroid associated orbitopathy.

Thyroid-associated orbitopathy (TAO) is a self-limiting auto-immune condition usually associated with Grave's disease. It is characterised by ocular pain, eyelid swelling, chemosis, proptosis and keratopathy. As the mechanism for ophthamoplegia and optic neuropathy is the orbital swelling leading to mechanical restriction of ocular muscles and compression of optic nerve, one expects proptosis rather than ptosis in TAO. We describe a case of a young adult woman who presented with acute onset restriction of movement along with partial ptosis and severe diminution of vision in left eye. The MRI of orbit revealed significant swelling of recti along with signal alteration consistent with TAO. The radio-isotope thyroid scan revealed thyroiditis, and thyroid peroxidase (TPO) antibody was significantly high; hence, the diagnosis of Hashimoto thyroiditis was considered. A course of intravenous methylprednisolone followed by oral steroid was administered, which produced marked improvement in vision and extraocular movement.

Freidreich's ataxia with retained reflexes: a phenotype and genotype correlation.

An 18-year-old lady had presented to us with insidious onset progressive gait ataxia of 5-year duration. Her sister had similar complaints and type 1 diabetes mellitus. Examination revealed, gait ataxia, impaired tandem gait, babinski sign and severe swaying on testing for Romberg's sign. All deep tendon reflexes were exaggerated. On investigations, there was no evidence for diabetes mellitus or nutritional deficiencies. Electrocardiogram and echocardiogram were normal. Magnetic spine resonance showed marked atrophy of cervical cord with normal cerebellum. The genetic testing disclosed expanded GAA repeat length on both alleles of FXN gene. The GAA repeat length on both alleles was much less than mean length observed in Friedreich's ataxia. This case highlights how strongly the genotype influences the neurological and systemic manifestations as well as severity of disease in Friedreich's ataxia.

An unusual case of POEMS syndrome.

We report a 48-year-old man, a farmer, presenting with chronic sensori-motor polyneuropathy. Electrophysiology revealed demyelinating type of neuropathy. Immunoelectrophoresis for monoclonal protein was negative; however, plasmacytoma was discovered on spine imaging. After receiving radiotherapy and chemotherapy over past 9 months there has been a considerable improvement in patient's disability. This case highlights the role of skeletal survey in evaluation of chronic demyelinating neuropathy.

Pathways to understanding mind: construct validity and predictive validity of maternal mind-mindedness.

The construct validity of maternal mind-mindedness (MM) was investigated in the context of its relations with children's later understanding of mind. MM measures were obtained from infant-mother (N = 52) interactions at 6 months, and from maternal interviews at 48 months. Children's understanding of mind was assessed using theory of mind (ToM) tasks at 45 and 48 months, and a stream of consciousness (SoC) task at 55 months. One of the early MM measures--mothers' appropriate mind-related comments--was a positive independent predictor of: (a) MM at 48 months, and (b) ToM and SoC performance at 45 to 55 months. Path analyses suggested direct links between mothers' use of appropriate mind-related comments and children's later understanding of mind.

Maternal mind-mindedness and attachment security as predictors of theory of mind understanding.

This study investigated relations between social interaction during infancy and children's subsequent theory of mind (ToM). Infant-mother pairs (N = 57) were observed in a free-play context at 6 months. Interactions were coded for (a) mothers' use of mental state language that commented appropriately on the infants' mental states, and (b) mothers' use of mental state language that did not appropriately reflect their infants' minds. A third variable was (c) security of attachment, which was assessed using the Strange Situation procedure at 12 months. Performance on a battery of ToM tasks at 45 and 48 months was positively correlated with (a), but was not related to (b) or (c). A regression analysis showed that mothers' use of appropriate mental state comments independently predicted overall ToM performance, accounting for 11% of the variance. Children's verbal ability was the only other independent predictor of ToM performance, accounting for 16% of the variance. These findings represent the earliest known social predictor of individual differences in ToM.