Enhancing cell-based therapies with synthetic gene circuits responsive to molecular stimuli.

Synthetic biology aims to contribute to the development of next-generation patient-specific cell-based therapies for chronic diseases especially through the construction of sophisticated synthetic gene switches to enhance the safety and spatiotemporal controllability of engineered cells. Indeed, switches that sense and process specific cues, which may be either externally administered triggers or endogenous disease-associated molecules, have emerged as powerful tools for programming and fine-tuning therapeutic outputs. Living engineered cells, often referred to as designer cells, incorporating such switches are delivered to patients either as encapsulated cell implants or by infusion, as in the case of the clinically approved CAR-T cell therapies. Here, we review recent developments in synthetic gene switches responsive to molecular stimuli, spanning regulatory mechanisms acting at the transcriptional, translational, and posttranslational levels. We also discuss current challenges facing clinical translation of cell-based therapies employing these devices.

Use of life cycle assessment for estimating impacts of waste-to-energy technologies in solid waste management systems: the case of Buenos Aires, Argentina.

Analysing municipal solid waste (MSW) management scenarios is relevant for planning future policies and actions toward a circular economy. Life cycle assessment (LCA) is appropriate for evaluating technologies of MSW treatment and their environmental impacts. However, in developing countries, advanced assessments are difficult to introduce due to the lack of technical knowledge, data and financial support. This research aims to assess the main potentialities of the introduction of waste-to-energy (WtE) systems in a developing Argentinean urban area considering the existing regulations about MSW recycling goals. The study was conducted with WRATE software and the proposed scenarios were current management, grate incineration of raw MSW and incineration of solid recovered fuel (SRF). In addition, a sensitivity analysis on the energy matrix was included. It was found that the production of SRF allows increasing the energy generation from waste by 200% and reducing the environmental impact of about 10% regarding the current MSW management system. Acidification Potential and Abiotic Depletion Potential were sensitive to changes in electricity mix. Results showed that if MSW reduction goals are achieved, changes in MSW composition will affect the performance of WtE plants and, in some cases, they will be not technically feasible. The outcomes of this study can be of interest for developing countries stakeholders and practitioners interested in LCA and sustainable MSW management.

Combinatorial protein dimerization enables precise multi-input synthetic computations.

Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein-protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.

Regulation of Transgene Expression by the Natural Sweetener Xylose.

Next-generation gene and engineered-cell therapies benefit from incorporating synthetic gene networks that can precisely regulate the therapeutic output in response to externally administered signal inputs that are safe, readily bioavailable and pleasant to take. To enable such therapeutic control, a mammalian gene switch is designed to be responsive to the natural sweetener xylose and its functionality is assessed in mouse studies. The gene switch consists of the bacterial transcription regulator XylR fused to a mammalian transactivator, which binds to an optimized promoter in the presence of xylose, thereby allowing dose-dependent transgene expression. The sensitivity of SWEET (sweetener-inducible expression of transgene) is improved by coexpressing a xylose transporter. Mice implanted with encapsulated SWEET-engineered cells show increased blood levels of cargo protein when taking xylose-sweetened water or coffee, or highly concentrated apple extract, while they do not respond to intake of a usual amount of carrots, which contain xylose. In a proof-of-concept therapeutic application study, type-1 diabetic mice engineered with insulin-expressing SWEET show lowered glycemia and increased insulin levels when administered this fairly diabetic-compliant sweetener, compared to untreated mice. A SWEET-based therapy appears to have the potential to integrate seamlessly into patients' life-style and food habits in the move toward personalized medicine.

Pneumothorax and/or Pneumomediastinum Worsens the Prognosis of COVID-19 Patients with Severe Acute Respiratory Failure: A Multicenter Retrospective Case-Control Study in the North-East of Italy.

Pneumothorax (PNX) and pneumomediastinum (PNM) are potential complications of COVID-19, but their influence on patients' outcomes remains unclear. The aim of the study was to assess incidence, risk factors, and outcomes of severe COVID-19 complicated with PNX/PNM. METHODS: A retrospective multicenter case-control analysis was conducted in COVID-19 patients admitted for respiratory failure in intermediate care units of the Treviso area, Italy, from March 2020 to April 2021. Clinical characteristics and outcomes of patients with and without PNX/PNM were compared. RESULTS: Among 1213 patients, PNX and/or PNM incidence was 4.5%. Among these, 42% had PNX and PNM, 33.5% only PNX, and 24.5% only PNM. COVID-19 patients with PNX/PNM showed higher in-hospital (p = 0.02) and 90-days mortality (p = 0.048), and longer hospitalization length (p = 0.002) than COVID-19 patients without PNX/PNM. At PNX/PNM occurrence, one-third of subjects was not mechanically ventilated, and the respiratory support was similar to the control group. PNX/PNM occurrence was associated with longer symptom length before hospital admission (p = 0.005) and lower levels of blood lymphocytes (p = 0.017). CONCLUSION: PNX/PNM are complications of COVID-19 associated with a worse prognosis in terms of mortality and length of hospitalization. Although they are more frequent in ventilated patients, they can occur in non-ventilated, suggesting that mechanisms other than barotrauma might contribute to their presentation.

Cell-Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes.

The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectively. A single aromatic amino acid decarboxylase is capable of converting both hydroxylated intermediates into the final neurotransmitter. The platform described here may facilitate future efforts to generate medically useful artificial cells and nanofactories.

Trabectedin Overrides Osteosarcoma Differentiative Block and Reprograms the Tumor Immune Environment Enabling Effective Combination with Immune Checkpoint Inhibitors.

Purpose: Osteosarcoma, the most common primary bone tumor, is characterized by an aggressive behavior with high tendency to develop lung metastases as well as by multiple genetic aberrations that have hindered the development of targeted therapies. New therapeutic approaches are urgently needed; however, novel combinations with immunotherapies and checkpoint inhibitors require suitable preclinical models with intact immune systems to be properly tested.Experimental Design: We have developed immunocompetent osteosarcoma models that grow orthotopically in the bone and spontaneously metastasize to the lungs, mimicking human osteosarcoma. These models have been used to test the efficacy of trabectedin, a chemotherapeutic drug utilized clinically for sarcomas and ovarian cancer.Results: Trabectedin, as monotherapy, significantly inhibited osteosarcoma primary tumor growth and lung metastases by both targeting neoplastic cells and reprogramming the tumor immune microenvironment. Specifically, trabectedin induced a striking differentiation of tumor cells by favoring the recruitment of Runx2, the master genetic regulator of osteoblastogenesis, on the promoter of genes involved in the physiologic process of terminal osteoblast differentiation. Differentiated neoplastic cells, as expected, showed reduced proliferation rate. Concomitantly, trabectedin enhanced the number of tumor-infiltrating T lymphocytes, with local CD8 T cells, however, likely post-activated or exhausted, as suggested by their high expression of the inhibitory checkpoint molecule PD-1. Accordingly, the combination with a PD-1-blocking antibody significantly increased trabectedin efficacy in controlling osteosarcoma progression.Conclusions: These results demonstrate the therapeutic efficacy of trabectedin in osteosarcoma treatment, unveiling its multiple activities and providing a solid rationale for its combination with immune checkpoint inhibitors. Clin Cancer Res; 23(17); 5149-61. ©2017 AACR.

PISMA: A Visual Representation of Motif Distribution in DNA Sequences.

BACKGROUND: Because the graphical presentation and analysis of motif distribution can provide insights for experimental hypothesis, PISMA aims at identifying motifs on DNA sequences, counting and showing them graphically. The motif length ranges from 2 to 10 bases, and the DNA sequences range up to 10 kb. The motif distribution is shown as a bar-code-like, as a gene-map-like, and as a transcript scheme. RESULTS: We obtained graphical schemes of the CpG site distribution from 91 human papillomavirus genomes. Also, we present 2 analyses: one of DNA motifs associated with either methylation-resistant or methylation-sensitive CpG islands and another analysis of motifs associated with exosome RNA secretion. AVAILABILITY AND IMPLEMENTATION: PISMA is developed in Java; it is executable in any type of hardware and in diverse operating systems. PISMA is freely available to noncommercial users. The English version and the User Manual are provided in Supplementary Files 1 and 2, and a Spanish version is available at www.biomedicas.unam.mx/wp-content/software/pisma.zip and www.biomedicas.unam.mx/wp-content/pdf/manual/pisma.pdf.

Endocrine changes in histiocytosis of the hypothalamic-pituitary axis.

Histiocytosis is characterized by proliferation of cells from the mononuclear phagocyte system, and may be divided into Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (including Erdheim-Chester disease [ECD]). While diabetes insipidus (DI) is the most common hypothalamic-pituitary consequence, anterior pituitary deficiencies are less known. This study analyzed the frequency and progression of pituitary hormone deficiencies and the radiographic findings in 9 patients (7 with LCH and 2 with ECD) with hypothalamic-pituitary (HP) axis. Eighty-nine percent of patients had DI (62% at diagnosis), and 78% had one or more anterior pituitary deficiencies (71% at diagnosis). HP involvement is relatively common in patients diagnosed with histiocytosis and hormone deficiencies may be present at diagnosis or appear gradually during the course of disease. Regular monitoring of these patients is recommended.

Multiple human papillomavirus infections are highly prevalent in the anal canal of human immunodeficiency virus-positive men who have sex with men.

BACKGROUND: Anal cancer has become one of the most common non-AIDS-defined tumors among Human Immunodeficiency Virus-positive (HIV+) individuals, and a rise in its incidence among HIV+ Men who have Sex with Men (MSM) has been shown, despite the introduction of Highly Active Anti-Retroviral Therapy (HAART). Human Papillomavirus (HPV) infections are highly prevalent among HIV+ MSM and recent studies have shown high rates of HPV-associated anal intraepithelial neoplasia (AIN) and anal cancer among this population. METHODS: In the present study we determined the prevalence and nature of HPV co-infections in the anal canal of 324 HIV+ MSM attending a high specialty medical center in Mexico City, DNA extraction and amplification with generic primers for HPV was performed, followed by detection of specific types and co-infections with INNO-Lipa, and identification of variants by amplification and sequencing of the E6 and LCR region of HPV 16. RESULTS: We found a very high prevalence of HPV infections among this cohort (86%), with more than one fourth of them (28%) positive for type 16. Among HPV16-positive patients, European variants were the most prevalent, followed by Asian-American ones. Among these individuals (HPV-16+), we identified co-infections with other 21 HPV types namely; 11, 51, 52, 6, 66, 68, 74, 18, 45, 35, 26, 44, 70, 53, 54, 82, 31, 33, 56, 58, 59. CONCLUSIONS: HIV+ MSM show a very high rate of HPV infections in the anal canal and those with type 16 exhibited a multiplicity of associated types. This study emphasizes the need for an early detection of HPV infections among HIV+ MSM in order to establish its utility to prevent anal neoplasia and cancer.

Analysis of CpG methylation sites and CGI among human papillomavirus DNA genomes.

BACKGROUND: The Human Papillomavirus (HPV) genome is divided into early and late coding sequences, including 8 open reading frames (ORFs) and a regulatory region (LCR). Viral gene expression may be regulated through epigenetic mechanisms, including cytosine methylation at CpG dinucleotides. We have analyzed the distribution of CpG sites and CpG islands/clusters (CGI) among 92 different HPV genomes grouped in function of their preferential tropism: cutaneous or mucosal. We calculated the proportion of CpG sites (PCS) for each ORF and calculated the expected CpG values for each viral type. RESULTS: CpGs are underrepresented in viral genomes. We found a positive correlation between CpG observed and expected values, with mucosal high-risk (HR) virus types showing the smallest O/E ratios. The ranges of the PCS were similar for most genomic regions except E4, where the majority of CpGs are found within islands/clusters. At least one CGI belongs to each E2/E4 region. We found positive correlations between PCS for each viral ORF when compared with the others, except for the LCR against four ORFs and E6 against three other ORFs. The distribution of CpG islands/clusters among HPV groups is heterogeneous and mucosal HR-HPV types exhibit both lower number and shorter island sizes compared to cutaneous and mucosal Low-risk (LR) HPVs (all of them significantly different). CONCLUSIONS: There is a difference between viral and cellular CpG underrepresentation. There are significant correlations between complete genome PCS and a lack of correlations between several genomic region pairs, especially those involving LCR and E6. L2 and L1 ORF behavior is opposite to that of oncogenes E6 and E7. The first pair possesses relatively low numbers of CpG sites clustered in CGIs while the oncogenes possess a relatively high number of CpG sites not associated to CGIs. In all HPVs, E2/E4 is the only region with at least one CGI and shows a higher content of CpG sites in every HPV type with an identified E4. The mucosal HR-HPVs show either the shortest CGI size, followed by the mucosal LR-HPVs and lastly by the cutaneous viral subgroup, and a trend to the lowest CGI number, followed by the cutaneous viral subgroup and lastly by the mucosal LR-HPVs.

Vaccines against human papillomavirus: perspectives for controlling cervical cancer.

Prophylactic vaccines against human papillomavirus (HPV) are on the market and will certainly reduce the incidence of genital warts and the risk of developing cervical cancer. In addition, they will contribute to reducing anal as well as head and neck cancers. However, effort should be made in the short term in order for these vaccines to have a real impact in the developing world, where almost 80% of cervical cancer cases occur. Since the available vaccines include only two of the HPV types found in cancers (approximately 70%), improvements in current mass screening programs - with the use of molecular techniques - must be made, particularly in developing countries. Therapeutic vaccines have been designed to control advanced lesions and residual illness and, although success has usually been obtained in animal models, clinical studies have not yet provided the anticipated results. Finally, the next generations of prophylactic HPV vaccines will probably include subunit vaccines, transgenic bacteria and plants, among others, and could represent useful and cheaper alternatives for reducing cervical cancer, particularly in the developing world.

[Phylogenetic distance between Giardia intestinalis isolates from symptomatic and asymptomatic children]. / Distancia filogenética de aislados de Giardia intestinalis de niños sintomáticos y asintomáticos.

The purpose of the present investigation, was to analyze the sequences of 16S ribosomal genes partially amplified from 17 isolated Giardia intestinalis obtained from faces of 13 children with intestinal pathology and four symptoms-free children with intestinal pathology. Analysis was made also with Giardia Portland-I and four Giardias isolated from dogs. Children ages in both groups ranged from six to twelve years. Genomic DNA was isolated using the phenol-chloroform-isoamyl alcohol technique, and partial amplification of 16S rRNA ribosomal gene was carried out by the Polymerase Chain Reaction. Sequences of rDNA were compared with Portland-I by using CLUSTAL-W (1.81) and PHYLIP (3.6) software in order to determine phylogenic associations. Our results showed that only one isolate from symptoms-free children and three from the symptomatic children were associated with Portland I. The other isolated Giardias were associated between them and with two samples obtained from dogs that are phylogenetically distant from Portland-I. Two isolates from dog constituted a different group. These results suggest a zoonotic infection and presence of symptoms in children from the present investigation; probably it was associated with host or special characteristics of Giardia strains. The last two situations have a special epidemiological and public health interest.

Distancia filogenética de aislados de Giardia intestinalis de niños sintomáticos y asintomáticos / Phylogenetic distance between Giardia intestinalis isolates from symptomatic and asymptomatic children

The purpose of the present investigation, was to analyze the sequences of 16S ribosomal genes partially amplified from 17 isolated Giardia intestinalis obtained from faces of 13 children with intestinal pathology and four symptoms-free children with intestinal pathology. Analysis was made also with Giardia Portland-I and four Giardias isolated from dogs. Children ages in both groups ranged from six to twelve years. Genomic DNA was isolated using the phenol-chloroform-isoamyl alcohol technique, and partial amplification of 16S rRNA ribosomal gene was carried out by the Polymerase Chain Reaction. Sequences of rDNA were compared with Portland-I by using CLUSTAL-W (1.81) and PHYLIP (3.6) software in order to determine phylogenic associations. Our results showed that only one isolate from symptoms-free children and three from the symptomatic children were associated with Portland I. The other isolated Giardias were associated between them and with two samples obtained from dogs that are phylogenetically distant from Portland-I. Two isolates from dog constituted a different group. These results suggest a zoonotic infection and presence of symptoms in children from the present investigation; probably it was associated with host or special characteristics of Giardia strains. The last two situations have a special epidemiological and public health interest.

Suplementación alimenticia por 9 meses con la papilla HIM-Maiz en niños desnutridos de comunidades rurales / Nutritional supplementation in malnourished children living in rural areas

Introducción. La suplementación alimentaria ha mostrado efectividad contra la desnutrición en áreas rurales. Objetivo. Evaluar el efecto de la ingestión por 9 meses de la papilla HIM-Maíz sobre el indicador peso/edad en desnutridos menores de 5 años. Material y métodos. El diseño del estudio fue experimental, prospectivo, longitudinal y de casos y controles. Se evaluó el efecto de la suplementación a la ingestión diaria, con la papilla HIM-Maíz por un período consecutivo de 9 meses (grupo estudio (GE)), sobre el estado nutricio (peso/edad) de niños desnutridos menores de 5 años en un municipio rural del estado de Hidalgo, en comparación con otro grupo de niños de las mismas características, de otro municipio, seguidos durante el mismo tiempo pero que no recibieron el suplemento (grupo control (GC)). Al GE se le realizarón 5 evaluaciones antropométricas, basal y 4 subsecuentes; al GC basal y final. Resultados. El GE menor o igual de 24 meses (n=40) mostró una Z basal de -2.5 y final de -1.93; el grupo mayor de 24 meses (n=51) -1.86 y -1.51 respectivamente. El GC iguales o menores de 24 meses (n=19) presentó Z basal de -1.72 y final de -1.76, el grupo mayor de 24 meses presentó Z -1.38. Conclusión. El suplemento papilla HIM-Maíz a los 9 y 12 meses de ingestión diaria, promueve el crecimiento en niños desnutridos de comunidades rurales