Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction.

The implementation and access to combined antiretroviral treatment (cART) have dramatically improved the quality of life of people living with HIV (PLWH). However, some comorbidities, such as neurological disorders associated with HIV infection still represent a serious clinical challenge. Soluble factors in plasma that are associated with control of HIV replication and neurological dysfunction could serve as early biomarkers and as new therapeutic targets for this comorbidity. We used a customized antibody array for determination of blood plasma factors in 40 untreated PLWH with different levels of viremia and found sirtuin-2 (SIRT2), an NAD-dependent deacetylase, to be strongly associated with elevated viral loads and HIV provirus levels, as well as with markers of neurological damage (a-synuclein [SNCA], brain-derived neurotrophic factor [BDNF], microtubule-associated protein tau [MAPT], and neurofilament light protein [NFL]). Also, longitudinal analysis in HIV-infected individuals with immediate (n = 9) or delayed initiation (n = 10) of cART revealed that after 1 year on cART, SIRT2 plasma levels differed between both groups and correlated inversely with brain orbitofrontal cortex involution. Furthermore, targeting SIRT2 with specific small-molecule inhibitors in in vitro systems using J-LAT A2 and primary glial cells led to diminished HIV replication and virus reactivation from latency. Our data thus identify SIRT2 as a novel biomarker of uncontrolled HIV infection, with potential impact on neurological dysfunction and offers a new therapeutic target for HIV treatment and cure. IMPORTANCE Neurocognitive disorders are frequently reported in people living with HIV (PLWH) even with the introduction of combined antiretroviral treatment (cART). To identify biomarkers and potential therapeutic tools to target HIV infection in peripheral blood and in the central nervous system (CNS), plasma proteomics were applied in untreated chronic HIV-infected individuals with different levels of virus control. High plasma levels of sirtuin-2 (SIRT2), an NAD+ deacetylase, were detected in uncontrolled HIV infection and were strongly associated with plasma viral load and proviral levels. In parallel, SIRT2 levels in the peripheral blood and CNS were associated with markers of neurological damage and brain involution and were more pronounced in individuals who initiated cART later in infection. In vitro infection experiments using specific SIRT2 inhibitors suggest that specific targeting of SIRT2 could offer new therapeutic treatment options for HIV infections and their associated neurological dysfunction.

Multidomain chimeric enzymes as a promising alternative for biocatalysts improvement: a minireview.

Searching for new and better biocatalysts is an area of study in constant development. In nature, mechanisms generally occurring in evolution, such as genetic duplication, recombination, and natural selection processes, produce various enzymes with different architectures and properties. The recombination of genes that code proteins produces multidomain chimeric enzymes that contain two or more domains that sometimes enhance their catalytic properties. Protein engineering has mimicked this process to enhance catalytic activity and the global stability of enzymes, searching for new and better biocatalysts. Here, we present and discuss examples from both natural and synthetic multidomain chimeric enzymes and how additional domains heighten their stability and catalytic activity. Moreover, we also describe progress in developing new biocatalysts using synthetic fusion enzymes and revise some methodological strategies to improve their biological fitness.

Adeno-Associated Viral Vectors as Versatile Tools for Parkinson's Research, Both for Disease Modeling Purposes and for Therapeutic Uses.

It is without any doubt that precision medicine therapeutic strategies targeting neurodegenerative disorders are currently witnessing the spectacular rise of newly designed approaches based on the use of viral vectors as Trojan horses for the controlled release of a given genetic payload. Among the different types of viral vectors, adeno-associated viruses (AAVs) rank as the ones most commonly used for the purposes of either disease modeling or for therapeutic strategies. Here, we reviewed the current literature dealing with the use of AAVs within the field of Parkinson's disease with the aim to provide neuroscientists with the advice and background required when facing a choice on which AAV might be best suited for addressing a given experimental challenge. Accordingly, here we will be summarizing some insights on different AAV serotypes, and which would be the most appropriate AAV delivery route. Next, the use of AAVs for modeling synucleinopathies is highlighted, providing potential readers with a landscape view of ongoing pre-clinical and clinical initiatives pushing forward AAV-based therapeutic approaches for Parkinson's disease and related synucleinopathies.

Recycling industrial wastewater for improved carbohydrate-rich biomass production in a semi-continuous photobioreactor: Effect of hydraulic retention time.

This study aimed to investigate a mixed microalgae culture's capacity to simultaneously remove nutrients and organic matter from industrial effluents while producing carbohydrate-rich biomass. A culture initially dominated by filamentous cyanobacteria Geitlerinema sp. was inoculated in a lab-scale stirred tank photobioreactor, operating at 10, 8, and 6 days hydraulic retention time (HRT). The results show that different HRT led to different inorganic carbon profiles and N:P ratios in the culture, influencing microbial changes, and carbohydrate content. Hence, higher N-NH4+ removal efficiencies were obtained at HRT of 10 d and decreased with decreasing HRT. Whereas, complete depletion of P-PO43- was achieved only at HRT of 8 d and 6 d. Also, the highest COD removal efficiency (60%) was achieved at 6 d of HRT. The maximum accumulation of carbohydrates was achieved at HRT of 8 d, which presented an N:P ratio of 22:1 and carbon availability, recording a constant carbohydrate content of 57% without any additional carbon source. Furthermore, this operational condition reached the best biomass production of 0.033 g L-1d-1 of easy-settling cyanobacteria dominated culture. According to the results, this process presents an alternative to recycling industrial effluents and, at the same time, grow valuable biomass, closing a loop for sustainable economy.

Vagus nerve dysfunction in the post-COVID-19 condition: a pilot cross-sectional study.

OBJECTIVES: The post-COVID-19 condition (PCC) is a disabling syndrome affecting at least 5%-10% of subjects who survive COVID-19. SARS-CoV-2 mediated vagus nerve dysfunction could explain some PCC symptoms, such as dysphonia, dysphagia, dyspnea, dizziness, tachycardia, orthostatic hypotension, gastrointestinal disturbances, or neurocognitive complaints. METHODS: We performed a cross-sectional pilot study in subjects with PCC with symptoms suggesting vagus nerve dysfunction (n = 30) and compared them with subjects fully recovered from acute COVID-19 (n = 14) and with individuals never infected (n = 16). We evaluated the structure and function of the vagus nerve and respiratory muscles. RESULTS: Participants were mostly women (24 of 30, 80%), and the median age was 44 years (interquartile range [IQR] 35-51 years). Their most prevalent symptoms were cognitive dysfunction 25 of 30 (83%), dyspnea 24 of 30 (80%), and tachycardia 24 of 30 (80%). Compared with COVID-19-recovered and uninfected controls, respectively, subjects with PCC were more likely to show thickening and hyperechogenic vagus nerve in neck ultrasounds (cross-sectional area [CSA] [mean ± standard deviation]: 2.4 ± 0.97mm2 vs. 2 ± 0.52mm2 vs. 1.9 ± 0.73 mm2; p 0.08), reduced esophageal-gastric-intestinal peristalsis (34% vs. 0% vs. 21%; p 0.02), gastroesophageal reflux (34% vs. 19% vs. 7%; p 0.13), and hiatal hernia (25% vs. 0% vs. 7%; p 0.05). Subjects with PCC showed flattening hemidiaphragms (47% vs. 6% vs. 14%; p 0.007), and reductions in maximum inspiratory pressure (62% vs. 6% vs. 17%; p ≤ 0.001), indicating respiratory muscle weakness. The latter findings suggest additional involvement of the phrenic nerve. DISCUSSION: Vagus and phrenic nerve dysfunction contribute to the complex and multifactorial pathophysiology of PCC.

Cultivation of carbohydrate-rich microalgae with great settling properties using cooling tower wastewater.

Wastewater treatment and simultaneous production of value-added products with microalgae represent a sustainable alternative. Industrial wastewater, characterized by high C/N molar ratios, can naturally improve the carbohydrate content in microalgae without the need for any external source of carbon while degrading the organic matter, macro-nutrients, and micro-nutrients. This study aimed to understand the treatment, reuse, and valorization mechanisms of real cooling tower wastewater (CWW) from a cement-processing industry mixed with domestic wastewater (DW) to produce microalgal biomass with potential for synthesis of biofuels or other value-added products. For this purpose, three photobioreactors with different hydraulic retention times (HRT) were inoculated simultaneously using the CWW-DW mixture. Macro- and micro-nutrient consumption and accumulation, organic matter removal, algae growth, and carbohydrate content were monitored for 55 days. High COD (> 80%) and macronutrient removals (> 80% of N and P) were achieved in all the photoreactors, with heavy metals below the limits established by local standards. The best results showed maximum algal growth of 1.02 g SSV L-1 and 54% carbohydrate accumulation with a C/N ratio of 31.24 mol mol-1. Additionally, the harvested biomass presented a high Ca and Si content, ranging from 11 to 26% and 2 to 4%, respectively. Remarkably, big flocs were produced during microalgae growth, which enhanced natural settling for easy biomass harvesting. Overall, this process represents a sustainable alternative for CWW treatment and valorization, as well as a green tool for generating carbohydrate-rich biomass with the potential to produce biofuels and fertilizers.

Adeno-Associated Viral Vectors as Versatile Tools for Neurological Disorders: Focus on Delivery Routes and Therapeutic Perspectives.

It is without doubt that the gene therapy field is currently in the spotlight for the development of new therapeutics targeting unmet medical needs. Thus, considering the gene therapy scenario, neurological diseases in general and neurodegenerative disorders in particular are emerging as the most appealing choices for new therapeutic arrivals intended to slow down, stop, or even revert the natural progressive course that characterizes most of these devastating neurodegenerative processes. Since an extensive coverage of all available literature is not feasible in practical terms, here emphasis was made in providing some advice to beginners in the field with a narrow focus on elucidating the best delivery route available for fulfilling any given AAV-based therapeutic approach. Furthermore, it is worth nothing that the number of ongoing clinical trials is increasing at a breath-taking speed. Accordingly, a landscape view of preclinical and clinical initiatives is also provided here in an attempt to best illustrate what is ongoing in this quickly expanding field.

Residual liver stiffness in the postpartum period in women with preeclampsia and healthy women: A case-control study.

OBJECTIVE: We aimed to assess the presence of residual liver stiffness in preeclampsia 45 days after delivery. STUDY DESIGN: This was a longitudinal case-control study to assess the association between residual liver stiffness, measured 45 days postpartum, and hypertensive disorders of pregnancy. Cases were obstetric patients admitted with hypertension during pregnancy (preeclampsia with and without severe features, or HELLP syndrome) between June 2019 and September 2021. Controls were obstetric patients with similar ages, social status, gestational age (assessed by the first-trimester ultrasound), and geographical areas between the same period. A 2:1 case-control match was considered. MAIN OUTCOME MEASURES: We recorded blood pressure and laboratory tests during the hospital stay. We measured the residual liver stiffness in preeclampsia participants and controls 45 days after delivery. RESULTS: We recruited 99 hypertensive patients and 46 control patients. The residual liver stiffness was higher in hypertensive cases than controls (4.6 [3.9-5.3] kPa vs. 4.0 [3.2-5.2] kPa; p = 0.039). Linear regression results showed that systolic and diastolic blood pressures and alanine aminotransferase positively correlated with liver stiffness values (p < 0.01). CONCLUSIONS: Preeclampsia and disease severity were associated with residual liver stiffness. Prospective studies should further evaluate the clinical implications of these findings.

Risk factors for SARS-CoV-2 transmission in close contacts of adults at high risk of infection due to occupation: results from the contact tracing strategy of the CoVIDA epidemiological surveillance study in Bogotá, Colombia, in 2020-2021.

OBJECTIVES: To estimate the risk factors for SARS-CoV-2 transmission in close contacts of adults at high risk of infection due to occupation, participants of the CoVIDA study, in Bogotá D.C., Colombia. SETTING: The CoVIDA study was the largest COVID-19 intensified sentinel epidemiological surveillance study in Colombia thus far, performing over 60 000 RT-PCR tests for SARS-CoV-2 infection. The study implemented a contact tracing strategy (via telephone call) to support traditional surveillance actions performed by the local health authority. PARTICIPANTS: Close contacts of participants from the CoVIDA study. PRIMARY AND SECONDARY OUTCOME MEASURES: SARS-CoV-2 testing results were obtained (RT-PCR with CoVIDA or self-reported results). The secondary attack rate (SAR) was calculated using contacts and primary cases features. RESULTS: The CoVIDA study performed 1257 contact tracing procedures on primary cases. A total of 5551 close contacts were identified and 1050 secondary cases (21.1%) were found. The highest SAR was found in close contacts: (1) who were spouses (SAR=32.7%; 95% CI 29.1% to 36.4%), (2) of informally employed or unemployed primary cases (SAR=29.1%; 95% CI 25.5% to 32.8%), (3) of symptomatic primary cases (SAR of 25.9%; 95% CI 24.0% to 27.9%) and (4) living in households with more than three people (SAR=22.2%; 95% CI 20.7% to 23.8%). The spouses (OR 3.85; 95% CI 2.60 to 5.70), relatives (OR 1.89; 95% CI 1.33 to 2.70) and close contacts of a symptomatic primary case (OR 1.48; 95% CI 1.24 to 1.77) had an increased risk of being secondary cases compared with non-relatives and close contacts of an asymptomatic index case, respectively. CONCLUSIONS: Contact tracing strategies must focus on households with socioeconomic vulnerabilities to guarantee isolation and testing to stop the spread of the disease.

Prediction of SARS-CoV-2 infection with a Symptoms-Based model to aid public health decision making in Latin America and other low and middle income settings.

Symptoms-based models for predicting SARS-CoV-2 infection may improve clinical decision-making and be an alternative to resource allocation in under-resourced settings. In this study we aimed to test a model based on symptoms to predict a positive test result for SARS-CoV-2 infection during the COVID-19 pandemic using logistic regression and a machine-learning approach, in Bogotá, Colombia. Participants from the CoVIDA project were included. A logistic regression using the model was chosen based on biological plausibility and the Akaike Information criterion. Also, we performed an analysis using machine learning with random forest, support vector machine, and extreme gradient boosting. The study included 58,577 participants with a positivity rate of 5.7%. The logistic regression showed that anosmia (aOR = 7.76, 95% CI [6.19, 9.73]), fever (aOR = 4.29, 95% CI [3.07, 6.02]), headache (aOR = 3.29, 95% CI [1.78, 6.07]), dry cough (aOR = 2.96, 95% CI [2.44, 3.58]), and fatigue (aOR = 1.93, 95% CI [1.57, 2.93]) were independently associated with SARS-CoV-2 infection. Our final model had an area under the curve of 0.73. The symptoms-based model correctly identified over 85% of participants. This model can be used to prioritize resource allocation related to COVID-19 diagnosis, to decide on early isolation, and contact-tracing strategies in individuals with a high probability of infection before receiving a confirmatory test result. This strategy has public health and clinical decision-making significance in low- and middle-income settings like Latin America.

Effectiveness and adherence to closed face shields in the prevention of COVID-19 transmission: a non-inferiority randomized controlled trial in a middle-income setting (COVPROSHIELD).

BACKGROUND: The use of respiratory devices can mitigate the spread of diseases such as COVID-19 in community settings. We aimed to determine the effectiveness of closed face shields with surgical face masks to prevent SARS-CoV-2 transmission in working adults during the COVID-19 pandemic in Bogotá, Colombia. METHODS: An open-label non-inferiority randomized controlled trial that randomly assigned participants to one of two groups: the intervention group was instructed to wear closed face shields with surgical face masks, and the active control group was instructed to wear only surgical face masks. The primary outcome was a positive reverse transcription polymerase chain reaction test, IgG/IgM antibody test for SARS-CoV-2 detection, or both during and at the end of the follow-up period of 21 days. The non-inferiority limit was established at - 5%. RESULTS: A total of 316 participants were randomized, 160 participants were assigned to the intervention group and 156 to the active control group. In total, 141 (88.1%) participants in the intervention group and 142 (91.0%) in the active control group completed the follow-up. PRIMARY OUTCOME: a positive SARS-CoV-2 test result was identified in one (0.71%) participant in the intervention group and three (2.1%) in the active control group. In the intention-to-treat analysis, the absolute risk difference was - 1.40% (95% CI [- 4.14%, 1.33%]), and in the per-protocol analysis, the risk difference was - 1.40% (95% CI [- 4.20, 1.40]), indicating non-inferiority of the closed face shield plus face mask (did not cross the non-inferiority limit). CONCLUSIONS: The use of closed face shields and surgical face masks was non-inferior to the surgical face mask alone in the prevention of SARS-CoV-2 infection in highly exposed groups. Settings with highly active viral transmission and conditions such as poor ventilation, crowding, and high mobility due to occupation may benefit from the combined use of masks and closed face shields to mitigate SARS-CoV-2 transmission. TRIAL REGISTRATION: ClinicalTrials.gov NCT04647305 . Registered on November 30, 2020.

Histamine promotes the expression of receptors TLR2 and TLR4 and amplifies sensitivity to lipopolysaccharide and lipoteichoic acid treatment in human gingival fibroblasts.

The vasoactive hydrophilic amine histamine is the most important molecule released by mast cells. However, histamine's role in activating intracellular responses in HGFs (human gingival fibroblasts) has not been evaluated to date. In the present study, we investigated the effect of histamine and of Gram-negative [LPS (lipopolysaccharide)] and Gram-positive [LTA (lipoteichoic acid)] bacterial components on the modulation of the inflammatory response of HGFs. We incubated HGFs with histamine to determine whether this hydrophilic amine regulates overexpression of TLR2 (Toll-like receptor 2) and TLR4, which recognize LTA and LPS respectively. Our experiments demonstrated that histamine increases transcription and translation of TLR2 and TLR4. Incubation with LTA or LPS in the presence of histamine markedly increased expression of COX2 (cyclo-oxygenase 2) and synthesis of prostaglandin E2. These results suggest that histamine plays an important role in modulating the innate immune response, and likewise, that LTA and LPS regulate the adaptive immune response. The present study provides information about the regulation and expression of molecules that promote chronic inflammatory processes leading to the emergence of periodontitis and the consequent loss of the dental organ.

Factors Associated With SARS-CoV-2 Infection in Bogotá, Colombia: Results From a Large Epidemiological Surveillance Study.

BACKGROUND: Epidemiologic surveillance of COVID-19 is essential to collect and analyse data to improve public health decision making during the pandemic. There are few initiatives led by public-private alliances in Colombia and Latin America. The CoVIDA project contributed with RT-PCR tests for SARS-CoV-2 in mild or asymptomatic populations in Bogotá. The present study aimed to determine the factors associated with SARS-CoV-2 infection in working adults. METHODS: COVID-19 intensified sentinel epidemiological surveillance study, from April 18, 2020, to March 29, 2021. The study included people aged 18 years or older without a history of COVID-19. Two main occupational groups were included: healthcare and essential services workers with high mobility in the city. Social, demographic, and health-related factors were collected via phone survey. Afterwards, the molecular test was conducted to detect SARS-CoV-2 infection. FINDINGS: From the 58,638 participants included in the study, 3,310 (5·6%) had a positive result. A positive result was associated with the age group (18-29 years) compared with participants aged 60 or older, participants living with more than three cohabitants, living with a confirmed case, having no affiliation to the health system compared to those with social health security, reporting a very low socioeconomic status compared to those with higher socioeconomic status, and having essential occupations compared to healthcare workers. INTERPRETATION: The CoVIDA study showed the importance of intensified epidemiological surveillance to identify groups with increased risk of infection. These groups should be prioritised in the screening, contact tracing, and vaccination strategies to mitigate the pandemic. FUNDING: The CoVIDA study was funded through donors managed by the philanthropy department of Universidad de los Andes.

Novel Nested Peptide Epitopes Recognized by CD4+ T Cells Induced by HIV-1 Conserved-Region Vaccines.

CD4+ T-cell responses play an important role in the immune control of the human immunodeficiency virus type 1 (HIV-1) infection and as such should be efficiently induced by vaccination. It follows that definition of HIV-1-derived peptides recognized by CD4+ T cells in association with HLA class II molecules will guide vaccine development. Here, we have characterized the fine specificity of CD4+ T cells elicited in human recipients of a candidate vaccine delivering conserved regions of HIV-1 proteins designated HIVconsv. The majority of these 19 most immunogenic regions contained novel epitopes, that is, epitopes not listed in the Los Alamos National Laboratory HIV Sequence Database, which were able in vitro to stimulate vaccinees' CD4+ T cells to proliferate and produce interferon-γ and tumor necrosis factor-α. Accumulation of HLA class II epitopes will eventually accelerate development of HIV-1 prophylactic and therapeutic vaccines.

Metered-dose inhalers versus nebulization for the delivery of albuterol for acute exacerbations of wheezing or asthma in children: A systematic review with meta-analysis.

OBJECTIVES: The benefits of metered-dose inhalers with a spacer (MDI+S) have increasingly been recognized as an alternative method of albuterol administration for treating pediatric asthma exacerbations. The aim of this systematic review was to compare the response to albuterol delivered through nebulization (NEB) with albuterol delivered through MDI+S in pediatric patients with asthma exacerbations. METHODS: We conducted an electronic search in MEDLINE/PubMed, EMBASE, Ovid, and ClinicalTrials. To be included in the review, a study had to a randomized clinical trial comparing albuterol delivered via NEB versus MDI+S; and had to report the rate of hospital admission (primary outcome), or any of the following secondary outcomes: oxygen arterial saturation, heart rate (HR), respiratory rate (RR), the pulmonary index score (PIS), adverse effects, and need for additional treatment. RESULTS: Fifteen studies (n = 2057) met inclusion criteria. No significant differences were found between the two albuterol delivery methods in terms of hospital admission (relative risk, 0.89; 95% confidence interval [CI], 0.55-1.46; I2 = 32%; p = .65). There was a significant reduction in the PIS score (mean difference [MD], -0.63; 95% CI, -0.91 to -0.35; I2 = 0%; p < .00001), and a significantly smaller increase in HR (better; MD -6.47; 95% CI, -11.69 to -1.25; I2 = 0%; p = .02) when albuterol was delivered through MDI+S than when it was delivered through NEB. CONCLUSIONS: This review, an update of a previously-published meta-analysis, showed a significant reduction in the PIS and a significantly smaller increase in HR when albuterol was delivered through MDI+S than when it was delivered through NEB.

Specificity of CD8+ T-Cell Responses Following Vaccination with Conserved Regions of HIV-1 in Nairobi, Kenya.

Sub-Saharan Africa carries the biggest burden of the human immunodeficiency virus type 1 (HIV-1)/AIDS epidemic and is in an urgent need of an effective vaccine. CD8+ T cells are an important component of the host immune response to HIV-1 and may need to be harnessed if a vaccine is to be effective. CD8+ T cells recognize human leukocyte antigen (HLA)-associated viral epitopes and the HLA alleles vary significantly among different ethnic groups. It follows that definition of HIV-1-derived peptides recognized by CD8+ T cells in the geographically relevant regions will critically guide vaccine development. Here, we study fine details of CD8+ T-cell responses elicited in HIV-1/2-uninfected individuals in Nairobi, Kenya, who received a candidate vaccine delivering conserved regions of HIV-1 proteins called HIVconsv. Using 10-day cell lines established by in vitro peptide restimulation of cryopreserved PBMC and stably HLA-transfected 721.221/C1R cell lines, we confirm experimentally many already defined epitopes, for a number of epitopes we define the restricting HLA molecule(s) and describe four novel HLA-epitope pairs. We also identify specific dominance patterns, a promiscuous T-cell epitope and a rescue of suboptimal T-cell epitope induction in vivo by its functional variant, which all together inform vaccine design.

Theta-phase closed-loop stimulation induces motor paradoxical responses in the rat model of Parkinson disease.

BACKGROUND: High-frequency deep brain stimulation (DBS) has become a widespread therapy used in the treatment of Parkinson's Disease (PD) and other diseases. Although it has proved beneficial, much recent attention has been centered around the potential of new closed-loop DBS implementations. OBJECTIVE: Here we present a new closed-loop DBS scheme based on the phase of the theta activity recorded from the motor cortex. By testing the implementation on freely moving 6-OHDA lesioned and control rats, we assessed the behavioral and neurophysiologic effects of this implementation and compared it against the classical high-frequency DBS. RESULTS: Results show that both stimulation modalities produce significant and opposite changes on the movement and neurophysiological activity. Close-loop stimulation, far from improving the animals' behavior, exert contrary effects to those of high-frequency DBS which reverts the parkinsonian symptoms. Motor improvement during open-loop, high-frequency DBS was accompanied by a reduction in the amount of cortical beta oscillations while akinetic and disturbed behavior during close-loop stimulation coincided with an increase in the amplitude of beta activity. CONCLUSION: Cortical-phase-dependent close-loop stimulation of the STN exerts significant behavioral and oscillatory changes in the rat model of PD. Open-loop and close-loop stimulation outcomes differed dramatically, thus suggesting that the scheme of stimulation determines the output of the modulation even if the target structure is maintained. The current framework could be extended in future studies to identify the correct parameters that would provide a suitable control signal to the system. It may well be that with other stimulation parameters, this sort of DBS could be beneficial.

CiPerGenesis, A Mutagenesis Approach that Produces Small Libraries of Circularly Permuted Proteins Randomly Opened at a Focused Region: Testing on the Green Fluorescent Protein.

Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134-H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.