Development of a conditional localization approach to control apicoplast protein trafficking in malaria parasites.

Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2-HCS1, we were able to control protein biotinylation in the apicoplast in a ligand-dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites.

Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress.

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCß domain within the CTD reveals an unusual double-layered ß-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the ß-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

A novel lipoate attachment enzyme is shared by Plasmodium and Chlamydia species.

Lipoate is an essential cofactor for enzymes that are important for central metabolism and other processes. In malaria parasites, scavenged lipoate from the human host is required for survival. The Plasmodium falciparum mitochondrion contains two enzymes (PfLipL1 and PfLipL2) that are responsible for activating mitochondrial proteins through the covalent attachment of lipoate (lipoylation). Lipoylation occurs via a novel redox-gated mechanism that remains poorly understood. We show that PfLipL1 functions as a redox switch that determines which downstream proteins will be activated. Based on the lipoate redox state, PfLipL1 either functions as a canonical lipoate ligase or as a lipoate activating enzyme which works in conjunction with PfLipL2. We demonstrate that PfLipL2 is a lipoyltransferase and is a member of a novel clade of lipoate attachment enzymes. We show that a LipL2 enzyme from Chlamydia trachomatis has similar activity, demonstrating conservation between intracellular pathogens from different phylogenetic kingdoms and supporting the hypothesis that an early ancestor of malaria parasites once contained a chlamydial endosymbiont. Redox-dependent lipoylation may regulate processes such as central metabolism and oxidative defense pathways.

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability.

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 µM to as low as 110 nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.

Crystal structure of lipoate-bound lipoate ligase 1, LipL1, from Plasmodium falciparum.

Plasmodium falciparum lipoate protein ligase 1 (PfLipL1) is an ATP-dependent ligase that belongs to the biotin/lipoate A/B protein ligase family (PFAM PF03099). PfLipL1 is the only known canonical lipoate ligase in Pf and functions as a redox switch between two lipoylation routes in the parasite mitochondrion. Here, we report the crystal structure of a deletion construct of PfLipL1 (PfLipL1Δ243-279 ) bound to lipoate, and validate the lipoylation activity of this construct in both an in vitro lipoylation assay and a cell-based lipoylation assay. This characterization represents the first step in understanding the redox dependence of the lipoylation mechanism in malaria parasites. Proteins 2017; 85:1777-1783. © 2017 Wiley Periodicals, Inc.

Risk factors and visual results in cases of LASIK flap repositioning due to folds or dislocation: case series and literature review.

The presence of a corneal flap is a hallmark of laser in situ keratomileusis (LASIK), which offers advantages in terms of speed of visual recovery; however, it also carries the risk of postoperative flap displacement. We conducted a retrospective review of all consecutive eyes on which LASIK was performed by one single surgeon at an ophthalmological institute in Colombia between May 2005 and January 2011, looking for eyes that required flap repositioning. Demographic data, preoperative refraction, hinge position, and visual outcomes following flap repositioning were evaluated. A literature review on the subject was also conducted. We found 37 eyes on which flap repositioning was performed-12 eyes (32.4 %) with subluxation and 25 eyes (67.6 %) with folds; 21 eyes (56.8 %) had a temporal hinge and 16 eyes (43.2 %) had a superior hinge. With regard to the total number of eyes on which LASIK was performed (2,595), the overall incidence was 1.4 %. Sixteen out of 2,093 eyes (0.8 %) with a superior hinge and 21 out of 502 eyes (4.2 %) with a temporal hinge had flap-related postoperative complications (p < 0.00). A final best-corrected visual acuity (BCVA) between 20/20 and 20/25 was found in 75.7 % and a final BCVA between 20/30 and 20/40 was found in 21.6 %. Only one eye had less than 20/40 (previous amblyopia). From the eight eyes with a BCVA between 20/30 and 20/40, three had residual microstriae and one had corneal haze. Six eyes (16.2 %) lost two or more lines of BCVA. Flap subluxation or folds requiring flap repositioning were significantly more frequent when a temporal hinge was used.

Energetics of zinc-mediated interactions in the allosteric pathways of metal sensor proteins.

A metal-mediated interprotomer hydrogen bond has been implicated in the allosteric mechanism of DNA operator binding in several metal-sensing proteins. Using computational methods, we investigate the energetics of such zinc-mediated interactions in members of the ArsR/SmtB family of proteins (CzrA, SmtB, CadC, and NmtR) and the MarR family zinc-uptake repressor AdcR, which feature similar interactions, but in sites that differ widely in their allosteric responsiveness. We provide novel structural insight into previously uncharacterized allosteric forms of these proteins using computational methodologies. We find this metal-mediated interaction to be significantly stronger (∼8 kcal/mol) at functional allosteric metal binding sites compared to a nonresponsive site (CadC) and the apo-proteins. Simulations of the apo-proteins further reveal that the high interaction energy works to overcome the considerable disorder at these hydrogen-bonding sites and functions as a "switch" to lock in a weak DNA-binding conformation once metal is bound. These findings suggest a conserved functional role of metal-mediated second coordination shell hydrogen bonds at allosterically responsive sites in zinc-sensing transcription regulators.

Solution NMR refinement of a metal ion bound protein using metal ion inclusive restrained molecular dynamics methods.

Correctly calculating the structure of metal coordination sites in a protein during the process of nuclear magnetic resonance (NMR) structure determination and refinement continues to be a challenging task. In this study, we present an accurate and convenient means by which to include metal ions in the NMR structure determination process using molecular dynamics (MD) simulations constrained by NMR-derived data to obtain a realistic and physically viable description of the metal binding site(s). This method provides the framework to accurately portray the metal ions and its binding residues in a pseudo-bond or dummy-cation like approach, and is validated by quantum mechanical/molecular mechanical (QM/MM) MD calculations constrained by NMR-derived data. To illustrate this approach, we refine the zinc coordination complex structure of the zinc sensing transcriptional repressor protein Staphylococcus aureus CzrA, generating over 130 ns of MD and QM/MM MD NMR-data compliant sampling. In addition to refining the first coordination shell structure of the Zn(II) ion, this protocol benefits from being performed in a periodically replicated solvation environment including long-range electrostatics. We determine that unrestrained (not based on NMR data) MD simulations correlated to the NMR data in a time-averaged ensemble. The accurate solution structure ensemble of the metal-bound protein accurately describes the role of conformational sampling in allosteric regulation of DNA binding by zinc and serves to validate our previous unrestrained MD simulations of CzrA. This methodology has potentially broad applicability in the structure determination of metal ion bound proteins, protein folding and metal template protein-design studies.

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii.

Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

Metal site occupancy and allosteric switching in bacterial metal sensor proteins.

All prokaryotes encode a panel of metal sensor or metalloregulatory proteins that govern the expression of genes that allows an organism to quickly adapt to toxicity or deprivation of both biologically essential transition metal ions, e.g., Zn, Cu, Fe, and heavy metal pollutants. As such, metal sensor proteins can be considered arbiters of intracellular transition metal bioavailability and thus potentially control the metallation state of the metalloproteins in the cell. Metal sensor proteins are specialized allosteric proteins that regulate transcription as a result direct binding of one or two cognate metal ions, to the exclusion of all others. In most cases, the binding of the cognate metal ion induces a structural change in a protein oligomer that either activates or inhibits operator DNA binding. A quantitative measure of the degree to which a particular metal drives metalloregulation of operator DNA-binding is the allosteric coupling free energy, ΔGc. In this review, we summarize recent work directed toward understanding metal occupancy and metal selectivity of these allosteric switches in selected families of metal sensor proteins and examine the structural origins of ΔGc in the functional context a thermodynamic "set-point" model of intracellular metal homeostasis.

Clinical differences between bipolar disorder and borderline personality disorder: a case report.

The clinical difference between bipolar disorder and borderline personality disorder has always been a diagnostic challenge, especially with type II bipolar disorder and subthreshold symptoms, opening a diagnostic bias with the consequent repercussions of inappropriate treatment. Both pathologies are often misdiagnosed initially. The objective of this article is to emphasise the main clinical differences between the two pathologies. We present the case of a patient with a long history of psychiatric symptoms that started in childhood, with considerable functional impairment, who met the criteria for both disorders, pointing to comorbidity. During follow-up, she responded favourably to psychotropic drugs, pushing the diagnosis towards the bipolar spectrum, due to the notable improvement. However, comorbidity should not be neglected due to its high presentation.

Crystal structure of the zinc-dependent MarR family transcriptional regulator AdcR in the Zn(II)-bound state.

Streptococcus pneumoniae adhesin competence regulator (AdcR), the first metal-dependent member of the multiple antibiotic resistance regulator (MarR) family of proteins, represses the transcription of a high-affinity zinc-specific uptake transporter, a group of surface antigen zinc-binding pneumococcal histidine triad proteins (PhtA, PhtB, PhtD, and PhtE), and an AdcA homologue (AdcAII). The 2.0 Å resolution structure of Zn(II)-bound AdcR reveals a highly helical two-fold-symmetric dimer with two distinct metal-binding sites per protomer. Zn(II) is tetrahedrally coordinated by E24, H42, H108, and H112 in what defines the primary sensing site in AdcR. Site 2 is a tetracoordinate site whose function is currently unknown. NMR methyl group perturbation experiments reveal that Zn(II) drives a global change in the structure of apo-AdcR that stabilizes a conformation that is compatible with DNA binding. This co-repression mechanism is unprecedented in MarR transcriptional regulators.

Clinical Differences between Bipolar Disorder and Borderline Personality Disorder: A Case Report. / Diferencias clínicas del trastorno bipolar y el trastorno límite de la personalidad: a propósito de un caso.

The clinical difference between bipolar disorder and borderline personality disorder has always been a diagnostic challenge, especially with type II bipolar disorder and subthreshold symptoms, opening a diagnostic bias with the consequent repercussions of inappropriate treatment. Both pathologies are often misdiagnosed initially. The objective of this article is to emphasise the main clinical differences between the two pathologies. We present the case of a patient with a long history of psychiatric symptoms that started in childhood, with considerable functional impairment, who met the criteria for both disorders, pointing to comorbidity. During follow-up, she responded favourably to psychotropic drugs, pushing the diagnosis towards the bipolar spectrum, due to the notable improvement. However, comorbidity should not be neglected due to its high presentation.

Efficacy and safety of weekly vitamin D3 in patients with fibromyalgia: 12-week, double-blind, randomized, controlled placebo trial.

INTRODUCTION: FM is a chronic musculoskeletal disorder characterized by the presence of generalized pain. There are contradictory results regarding the prevalence and supplementation effect of vitamin D deficiency on FM patients. We aim to determine the safety and efficacy of a 12-week vitamin D supplementation on FM patients. METHODS: We conducted a randomized, placebo-controlled, double-blind clinical trial. We included female participants of 18 years old or older, who met 1990 or 2010 ACR criteria for fibromyalgia. The Spanish validated FIQ and the VAS of pain were applied at baseline. The participants were then randomized to receive placebo or 50,000 IU of Vitamin D3 PO, weekly for 12 weeks. RESULTS: We included 80 patients. There was no statistical difference in the initial and final FIQ between both groups. The FIQ delta also did not prove to be different at the end of the study. The increase in vitamin D levels in the intervention group was corroborated. Regarding serious adverse effects, none was reported in both groups. There was no statistical difference in minor adverse events. CONCLUSION: In this double-blind placebo-controlled randomized study conducted to measure the efficacy and safety of vitamin D exclusively in patients with FM, we found that there is no evidence of a trend in favor of vitamin D treatment, since we did not observe improvement in the VAS of pain or FIQ. TRIAL REGISTRY: Clinical Trials.gov number: NCT03369379 Key Points • There are conflicting results in vitamin D to treat fibromyalgia. • In this double-blind, randomized controlled trial, we did not find a difference in the VAS nor FIQ with vitamin D supplementation. • The increase in vitamin D levels in the intervention group was corroborated.

Discovery and Optimization of Triazine Nitrile Inhibitors of Toxoplasma gondii Cathepsin L for the Potential Treatment of Chronic Toxoplasmosis in the CNS.

With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains one of the most pervasive and infectious parasites. Toxoplasma infection is the second leading cause of death due to foodborne illness in the United States, causes severe disease in immunocompromised patients, and is correlated with several cognitive and neurological disorders. Currently, no therapies exist that are capable of eliminating the persistent infection in the central nervous system (CNS). In this study we report the identification of triazine nitrile inhibitors of Toxoplasma cathepsin L (TgCPL) from a high throughput screen and their subsequent optimization. Through rational design, we improved inhibitor potency to as low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold for TgCPL versus human isoform), and improved metabolic stability (t1/2 > 60 min in mouse liver microsomes) guided by a metabolite ID study. We demonstrated that this class of compounds is capable of crossing the blood-brain barrier in mice (1:1 brain/plasma at 2 h). Importantly, we also show for the first time that treatment of T. gondii bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite viability with efficacy equivalent to a TgCPL genetic knockout.

mSphere of Influence: Ushering in the CRISPR Revolution to Toxoplasma Biology.

Alfredo J. Guerra works in the field of molecular parasitology and structural biology. In this mSphere of Influence article, he reflects on how "Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9" by Bang Shen et al. (mBio 5:e01114-14, 2014, https://doi.org/10.1128/mBio.01114-14) and "Efficient Genome Engineering of Toxoplasma gondii using CRISPR/CAS9" by Saima M. Sidik et al. (PLoS One 9:e100450, 2014, https://doi.org/10.1371/journal.pone.0100450) made an impact on him by successfully implementing strategies to genetically manipulate T. gondii using CRISPR/CAS9 gene editing technology.

Safety and Therapeutic Profile of a GnRH-Based Vaccine Candidate Directed to Prostate Cancer. A 10-Year Follow-Up of Patients Vaccinated With Heberprovac.

Heberprovac is a GnRH based vaccine candidate containing 2.4 mg of the GnRHm1-TT peptide as the main active principle; 245 µg of the very small size proteoliposomes adjuvant (VSSP); and 350 µL of Montanide ISA 51 VG oil adjuvant. The aim of this study was to assess the safety and tolerance of the Heberprovac in advanced prostate cancer patients as well as its capacity to induce anti-GnRH antibodies, the subsequent effects on serum levels of testosterone and PSA and the patient overall survival. The study included eight patients with histologically-proven advanced prostate cancer with indication for hormonal therapy, who received seven intramuscular immunizations with Heberprovac within 18 weeks. Anti-GnRH antibody titers, testosterone and PSA levels, as well as clinical parameters were recorded and evaluated. The vaccine was well tolerated. Significant reductions in serum levels of testosterone and PSA were seen after four immunizations. Castrate levels of testosterone were observed in all patients at the end of the immunization schedule, which remained at the lowest level for at least 20 months. In a 10-year follow-up three out of six patients who completed the entire trial survived. In contrast only one out eight patients survived in the same period in a matched randomly selected group receiving standard anti-hormonal treatment. Heberprovac vaccination showed a good security profile, as well as immunological, biochemical and, most importantly, clinical benefit. The vaccinated group displayed survival advantage compared with the reference group that received standard treatment. These results warrant further clinical trials with Heberprovac involving a larger cohort.

Manejo conservador del neumotórax oculto en trauma

Introducción: El neumotórax oculto (NTXO) se encuentra hasta en el 15% de los traumatismos torácicos. Existen antecedentes del manejo conservador de esta patología (sólo observación), aunque su práctica continúa siendo discutida, especialmente, en traumatismos penetrantes. El objetivo de este trabajo es describir nuestra experiencia en el manejo conservador del NTXO. Materiales y Método: Estudio de cohorte retrospectivo realizado durante un período de 3 años en un Hospital de Trauma nivel I. Se incluyeron pacientes con traumatismo torácico (cerrado o penetrante) con NTXO. Se dividieron en dos grupos (conservados o drenados), realizándose una comparación de su evolución. Resultados: En 3 años fueron admitidos con traumatismo torácico 679 pacientes. De 93 pacientes con NTXO, 74 (80%) fueron conservados inicialmente y 19 (20%) tratados con drenaje pleural. Dos (3%) presentaron progresión del neumotórax en el seguimiento radiológico (conservación fallida). No se registraron complicaciones relacionadas con la ausencia de drenaje pleural. Las complicaciones y estancia hospitalaria fueron menores en el grupo de manejo conservador. Conclusión: Pacientes con NTXO por traumatismo de tórax (cerrado o penetrante), sin requerimiento de ventilación asistida y hemodinámicamente estables, pueden manejarse de manera conservadora con un monitoreo cercano durante 24 horas en forma segura, con menor tasa de complicaciones y de estancia hospitalaria.

Background: Occult pneumothorax (OPTX) is found in up to 15% of chest injuries. There is a history of conservative management of this pathology (only observation), although its practice continues to be discussed, especially in penetrating trauma. The objective of this paper is to describe our experience in the conservative management of OPTX. Materials and Method: Retrospective cohort study conducted over a 3-year period at a level I Trauma Center. Patients with thoracic trauma (blunt or penetrating) with OPTX were included. They were divided into two groups (preserved or drained) comparing their evolution. Results: Over a 3-year period 679 patients were admitted with chest trauma. From 93 patients with OPTX, 74 (80%) were initially preserved and 19 (20%) drained. Two patients (3%) presented pneumothorax progression in the follow-up imaging. There were no complications related to the absence of pleural drainage. Complications and hospital stay were lower in the conservative management group. Conclusion: Patients with OPTX due to chest trauma (blunt or penetrating), without requiring assisted ventilation and hemodynamically stable, can be safely conservative managed with close monitoring for 24 hours, with a lower rate of complications and hospital stay.

Structural Features of Apicomplexan Pore-Forming Proteins and Their Roles in Parasite Cell Traversal and Egress.

Apicomplexan parasites cause diseases, including malaria and toxoplasmosis, in a range of hosts, including humans. These intracellular parasites utilize pore-forming proteins that disrupt host cell membranes to either traverse host cells while migrating through tissues or egress from the parasite-containing vacuole after replication. This review highlights recent insight gained from the newly available three-dimensional structures of several known or putative apicomplexan pore-forming proteins that contribute to cell traversal or egress. These new structural advances suggest that parasite pore-forming proteins use distinct mechanisms to disrupt host cell membranes at multiple steps in parasite life cycles. How proteolytic processing, secretion, environment, and the accessibility of lipid receptors regulate the membranolytic activities of such proteins is also discussed.