Bioengineered Salivary Gland Microtissues─A Review of 3D Cellular Models and their Applications.

Salivary glands (SGs) play a vital role in maintaining oral health through the production and release of saliva. Injury to SGs can lead to gland hypofunction and a decrease in saliva secretion manifesting as xerostomia. While symptomatic treatments for xerostomia exist, effective permanent solutions are still lacking, emphasizing the need for innovative approaches. Significant progress has been made in the field of three-dimensional (3D) SG bioengineering for applications in gland regeneration. This has been achieved through a major focus on cell culture techniques, including soluble cues and biomaterial components of the 3D niche. Cells derived from both adult and embryonic SGs have highlighted key in vitro characteristics of SG 3D models. While still in its first decade of exploration, SG spheroids and organoids have so far served as crucial tools to study SG pathophysiology. This review, based on a literature search over the past decade, covers the importance of SG cell types in the realm of their isolation, sourcing, and culture conditions that modulate the 3D microenvironment. We discuss different biomaterials employed for SG culture and the current advances made in bioengineering SG models using them. The success of these 3D cellular models are further evaluated in the context of their applications in organ transplantation and in vitro disease modeling.

Internally bridged nanosilica for loadings and release of sparsely soluble compounds.

The engineering of a new monodisperse colloid with a sea urchin-like structure with a large complex internal structure is reported, in which silica surfaces are bridged by an aromatic organic cross-linker to serve as a nanocarrier host for drugs such as doxorubicin (DOX) against breast cancer cells. While dendritic fibrous nanosilica (DFNS) was employed and we do not observe a dendritic structure, these particles are referred to as sea urchin-like nanostructured silica (SNS). Since the structure of SNS consists of many silica fibrils protruding from the core, similar to the hairs of a sea urchin. For the aromatic structured cross-linker, bis(propyliminomethyl)benzene (b(PIM)B-S or silanated terephtaldehyde) were employed, which are prepared with terephtaldehyde and 3-aminopropyltriethoxy-silane (APTES) through a simple Schiff base reaction. b(PIM)B-S bridges were introduced into SNS under open vessel reflux conditions. SPS refers to the product obtained by incorporating the cross-linker b(PIM)B-S in ultra-small colloidal SNS particles. In-situ incorporation of DOX molecules resulted in SPS-DOX. The pH-responsive SPS nanocomposites were tested as biocompatible nanocarriers for controllable doxorubicin (DOX) delivery. We conclude that SPS is a unique colloid which has promising potential for technological applications such as advanced drug delivery systems, wastewater remediation and as a catalyst for green organic reactions in water.

Salivary gland bioengineering - yesterday, today, tomorrow!

Salivary glands are specialized structures developed as an extensively compact, arborized design through classical embryogenesis, accompanied by a cascade of events channelized by numerous growth factors and genetic regulatory pathways. Salivary secretions maintain oral homeostasis and, when diminished in certain conditions, present as xerostomia or salivary hypofunction, adversely impacting the patient's quality of life. The current available treatments primarily aim at tackling the immediate symptoms providing temporary relief to the patient. Despite scientific efforts to develop permanent and effective solutions to restore salivation, a significant permanent treatment is yet to be established. Tissue engineering has proven as a promising remedial tool in several diseases, as well as in xerostomia, and aims to restore partial loss of organ function. Recapitulating the physiological cellular microenvironment to in vitro culture conditions is constantly evolving. Replicating the dynamic multicellular interactions, genetic pathways, and cytomorphogenic forces, as displayed during salivary gland development have experienced considerable barriers. Through this review, we endeavour to provide an outlook on the evolution of in vitro salivary gland research, highlighting the key bioengineering advances and the challenges faced with the current therapeutic strategies for salivary hypofunction, with an insight into our team's scientific contributions.

Hydrogels for Salivary Gland Tissue Engineering.

Mimicking the complex architecture of salivary glands (SGs) outside their native niche is challenging due their multicellular and highly branched organization. However, significant progress has been made to recapitulate the gland structure and function using several in vitro and ex vivo models. Hydrogels are polymers with the potential to retain a large volume of water inside their three-dimensional structure, thus simulating extracellular matrix properties that are essential for the cell and tissue integrity. Hydrogel-based culture of SG cells has seen a tremendous success in terms of developing platforms for cell expansion, building an artificial gland, and for use in transplantation to rescue loss of SG function. Both natural and synthetic hydrogels have been used widely in SG tissue engineering applications owing to their properties that support the proliferation, reorganization, and polarization of SG epithelial cells. While recent improvements in hydrogel properties are essential to establish more sophisticated models, the emphasis should still be made towards supporting factors such as mechanotransduction and associated signaling cues. In this concise review, we discuss considerations of an ideal hydrogel-based biomaterial for SG engineering and their associated signaling pathways. We also discuss the current advances made in natural and synthetic hydrogels for SG tissue engineering applications.

Preparation of vibrational quasi-bound states of the transition state complex BrHBr from the bihalide ion BrHBr.

Efficient strategies that allow the preparation of molecular systems in particular vibrational states are important in the application of quantum control schemes to chemical reactions. In this paper, we propose the preparation of quasi-bound vibrational states of the collinear transition state complex BrHBr, from vibrational states of the bihalide ion BrHBr-, that favor the bond selective breakage of BrHBr. The results shown complement the investigation that we reported in a previous paper, [A. J. Garzón-Ramírez, J. G. López and C. A. Arango, Int. J. Quantum Chem., 2018, 24, e25784], in which we demonstrated the feasibility of controlling the bond selective decomposition of the collinear BrHBr using linear combinations of reactive resonances. We employed a dipole moment surface, calculated at the QCISD/d-aug-cc-pVTZ level of theory, to simulate the interaction of the BrHBr- ground vibrational state with heuristically optimized sequences of ultrashort infrared linear chirped laser pulses to achieve a target vibrational state, resulting from expanding a chosen linear combination of reactive resonances of BrHBr in terms of vibrational eigenstates of BrHBr-. The results of our simulations show final states that capture the most relevant features of the target state with different levels of description depending on the sequence of laser pulses employed. We also discuss ways of improving the description of the target state and possible limitations of our approach.

Polymeric Scaffolds for Dental, Oral, and Craniofacial Regenerative Medicine.

Dental, oral, and craniofacial (DOC) regenerative medicine aims to repair or regenerate DOC tissues including teeth, dental pulp, periodontal tissues, salivary gland, temporomandibular joint (TMJ), hard (bone, cartilage), and soft (muscle, nerve, skin) tissues of the craniofacial complex. Polymeric materials have a broad range of applications in biomedical engineering and regenerative medicine functioning as tissue engineering scaffolds, carriers for cell-based therapies, and biomedical devices for delivery of drugs and biologics. The focus of this review is to discuss the properties and clinical indications of polymeric scaffold materials and extracellular matrix technologies for DOC regenerative medicine. More specifically, this review outlines the key properties, advantages and drawbacks of natural polymers including alginate, cellulose, chitosan, silk, collagen, gelatin, fibrin, laminin, decellularized extracellular matrix, and hyaluronic acid, as well as synthetic polymers including polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly (ethylene glycol) (PEG), and Zwitterionic polymers. This review highlights key clinical applications of polymeric scaffolding materials to repair and/or regenerate various DOC tissues. Particularly, polymeric materials used in clinical procedures are discussed including alveolar ridge preservation, vertical and horizontal ridge augmentation, maxillary sinus augmentation, TMJ reconstruction, periodontal regeneration, periodontal/peri-implant plastic surgery, regenerative endodontics. In addition, polymeric scaffolds application in whole tooth and salivary gland regeneration are discussed.

Decellularized Extracellular Matrix Composite Hydrogel Bioinks for the Development of 3D Bioprinted Head and Neck in Vitro Tumor Models.

Reinforced extracellular matrix (ECM)-based hydrogels recapitulate several mechanical and biochemical features found in the tumor microenvironment (TME) in vivo. While these gels retain several critical structural and bioactive molecules that promote cell-matrix interactivity, their mechanical properties tend toward the viscous regime limiting their ability to retain ordered structural characteristics when considered as architectured scaffolds. To overcome this limitation characteristic of pure ECM hydrogels, we present a composite material containing alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, as rheological modifiers which impart mechanical integrity to the biologically active decellularized ECM (dECM). After an optimization process, the reinforced gel proposed is mechanically stable and bioprintable and has a stiffness within the expected physiological values. Our hydrogel's elastic modulus has no significant difference when compared to tumors induced in preclinical xenograft head and neck squamous cell carcinoma (HNSCC) mouse models. The bioprinted cell-laden model is highly reproducible and allows proliferation and reorganization of HNSCC cells while maintaining cell viability above 90% for periods of nearly 3 weeks. Cells encapsulated in our bioink produce spheroids of at least 3000 µm2 of cross-sectional area by day 15 of culture and are positive for cytokeratin in immunofluorescence quantification, a common marker of HNSCC model validation in 2D and 3D models. We use this in vitro model system to evaluate the standard-of-care small molecule therapeutics used to treat HNSCC clinically and report a 4-fold increase in the IC50 of cisplatin and an 80-fold increase for 5-fluorouracil compared to monolayer cultures. Our work suggests that fabricating in vitro models using reinforced dECM provides a physiologically relevant system to evaluate malignant neoplastic phenomena in vitro due to the physical and biological features replicated from the source tissue microenvironment.

Alginate-gelatin-Matrigel hydrogels enable the development and multigenerational passaging of patient-derived 3D bioprinted cancer spheroid models.

Hydrogels consisting of controlled fractions of alginate, gelatin, and Matrigel enable the development of patient-derived bioprinted tissue models that support cancer spheroid growth and expansion. These engineered models can be dissociated to be then reintroduced to new hydrogel solutions and subsequently reprinted to generate multigenerational models. The process of harvesting cells from 3D bioprinted models is possible by chelating the ions that crosslink alginate, causing the gel to weaken. Inclusion of the gelatin and Matrigel fractions to the hydrogel increases the bioactivity by providing cell-matrix binding sites and promoting cross-talk between cancer cells and their microenvironment. Here we show that immortalized triple-negative breast cancer cells (MDA-MB-231) and patient-derived gastric adenocarcinoma cells can be reprinted for at least three 21 d culture cycles following bioprinting in the alginate/gelatin/Matrigel hydrogels. Our drug testing results suggest that our 3D bioprinted model can also be used to recapitulatein vivopatient drug response. Furthermore, our results show that iterative bioprinting techniques coupled with alginate biomaterials can be used to maintain and expand patient-derived cancer spheroid cultures for extended periods without compromising cell viability, altering division rates, or disrupting cancer spheroid formation.

Egg White Alginate as a Novel Scaffold Biomaterial for 3D Salivary Cell Culturing.

Saliva production by salivary glands play a crucial role in oral health. The loss of salivary gland function could lead to xerostomia, a condition also known as dry mouth. Significant reduction in saliva production could lead to further complications such as difficulty in speech, mastication, and increased susceptibility to dental caries and oral infections and diseases. While some palliative treatments are available for xerostomia, there are no curative treatments to date. This study explores the use of Egg White Alginate (EWA), as an alternative scaffold to Matrigel® for culturing 3D salivary gland cells. A protocol for an optimized EWA was established by comparing cell viability using 1%, 2%, and 3% alginate solution. The normal salivary simian virus 40-immortalized acinar cell (NS-SV-AC) and the submandibular gland-human-1 (SMG-hu-1) cell lines were also used to compare the spheroid formation and cell viability properties of both scaffold biomaterials; cell viability was observed over 10 days using a Live-Dead Cell Assay. Cell viability and spheroid size in 2% EWA was significantly greater than 1% and 3%. It is evident that EWA can support salivary cell survivability as well as form larger spheroids when compared to cells grown in Matrigel®. However, further investigations are necessary as it is unclear if cultured cells were proliferating or aggregating.

Biotemplated Hollow Mesoporous Silica Particles as Efficient Carriers for Drug Delivery.

We designed three types of hollow-shaped porous silica materials via a three-step biotemplate-directed method: porous hollow silica nanorods, hollow dendritic fibrous nanostructured silica (DFNS), and ultraporous sponge-like DFNS. The first step was making a biotemplate, for which we used cellulose nanocrystals (CNCs), consisting of rod-shaped nanoparticles synthesized by conventional acid hydrolysis of cellulose fibers. In a second step, core-shell samples were prepared using CNC particles as hard template by two procedures. In the first one, core-shell CNC-silica nanoparticles were synthesized by a polycondensation reaction, which exclusively took place at the surface of the CNCs. In the second procedure, a typical synthesis of DFNS was conducted in a bicontinuous microemulsion with the assistance of additives. DFNS was assembled on the surface of the CNCs, giving rise to core-shell CNC-DFNS structures. Finally, all of the silica-coated CNC composites were calcined, during which the CNC was removed from the core and hollow structures were formed. These materials are very lightweight and highly porous. All three structures were tested as nanocarriers for drug delivery and absorbents for dye removal applications. Dye removal results showed that they can adsorb methylene blue efficiently, with ultraporous sponge-like DFNS showing the highest adsorption capacity, followed by hollow DFNS and hollow silica nanorods. Furthermore, breast cancer cells show a lower cell viability when exposed to doxorubicin-loaded hollow silica nanorods compared with control or doxorubicin cultures, suggesting that the loaded nanorod has a greater anticancer effect than free doxorubicin.

The Optimization of a Novel Hydrogel-Egg White-Alginate for 2.5D Tissue Engineering of Salivary Spheroid-Like Structure.

Hydrogels have been used for a variety of biomedical applications; in tissue engineering, they are commonly used as scaffolds to cultivate cells in a three-dimensional (3D) environment allowing the formation of organoids or cellular spheroids. Egg white-alginate (EWA) is a novel hydrogel which combines the advantages of both egg white and alginate; the egg white material provides extracellular matrix (ECM)-like proteins that can mimic the ECM microenvironment, while alginate can be tuned mechanically through its ionic crosslinking property to modify the scaffold's porosity, strength, and stiffness. In this study, a frozen calcium chloride (CaCl2) disk technique to homogenously crosslink alginate and egg white hydrogel is presented for 2.5D culture of human salivary cells. Different EWA formulations were prepared and biologically evaluated as a spheroid-like structure platform. Although all five EWA hydrogels showed biocompatibility, the EWA with 1.5% alginate presented the highest cell viability, while EWA with 3% alginate promoted the formation of larger size salivary spheroid-like structures. Our EWA hydrogel has the potential to be an alternative 3D culture scaffold that can be used for studies on drug-screening, cell migration, or as an in vitro disease model. In addition, EWA can be used as a potential source for cell transplantation (i.e., using this platform as an ex vivo environment for cell expansion). The low cost of producing EWA is an added advantage.

Highly Absorbent Antibacterial and Biofilm-Disrupting Hydrogels from Cellulose for Wound Dressing Applications.

In this study, a carboxyl-modified cellulosic hydrogel was developed as the base material for wound dressings. ε-poly-l-lysine, a natural polyamide, was then covalently linked to the hydrogel through a bioconjugation reaction, which was confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR). The antibacterial efficacy of the hydrogel was tested against two model bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, two of the most commonly found bacteria in wound infections. Bacterial viability and biofilm formation after exposure of bacteria to the hydrogels were used as efficacy indicators. Live/Dead assay was used to measure the number of compromised bacteria using a confocal laser scanning microscope. The results show that the antibacterial hydrogel was able to kill approximately 99% of the exposed bacteria after 3 h of exposure. In addition, NIH/3T3 fibroblasts were used to study the biocompatibility of the developed hydrogels. Water-soluble tetrazolium salt (WST)-1 assay was used to measure the metabolic activity of the cells and Live/Dead assay was used to measure the viability of the cells after 24, 48, and 72 h. The developed antibacterial hydrogels are light weight, have a high water-uptake capacity, and show high biocompatibility with the model mammalian cells, which make them a promising candidate to be used for wound dressing applications.

Management of Fertigation in Horticultural Crops through Automation with Electrotensiometers: Effect on the Productivity of Water and Nutrients.

Water and nutrient requirements of horticultural crops are influenced by different factors such as: Type of crop, stage of development and production system. Although greenhouse horticultural crops are more efficient in the use of water and fertilizers compared to other production systems, it is necessary increase efficiency for which individualized fertigation strategies must be designed for each greenhouse. The automation of fertigation based on the level of soil moisture allows optimization of management. The objective of this work was to determine the influence of the activation command of fertigation with electrotensiometers and the characteristics of the greenhouse on the productivity of the crop and the efficiency of use of water and nutrients in a sweet pepper crop. The trial was developed in two greenhouses. Four treatments were studied, combination of who two-factor: Soil matric potential (SMP) (SMP-10: Automatic activation of irrigation to -10 kPa and SMP-20: Automatic activation of irrigation to -20 kPa) and greenhouse characteristics (G1 and G2). The nutritive solution applied was the same in all treatments. The yield and volume of water and nutrients applied were determined, calculating the productivity of the water (WP), as well as productivity the nutrients. The fertigation activation threshold of -10 kPa presented the best results, increasing the yield and conserving WP and nutrient productivity with respect to -20 kPa in both greenhouses. The automation of irrigation with electrotensiometers allowed the application of different volume of fertigation demanded by the crop in each greenhouse, equalizing the WP and nutrient productivity without producing drainage. The pepper crop in the greenhouse G1 presented greater vegetative development, higher yield and demanded a greater volume of fertigation than G2 regardless of the activation threshold. This was due to the fact that the soil matric potential after irrigation in greenhouse G1 was closer to zero, being able to conclude that not only the soil matric potential threshold of irrigation activation has an influence on crop, but also the potential registered after irrigation. Soil matric potentials closer to zero are more productive.

Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics.

Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components. The composite gels are demonstrated as a cell-laden bioink to build 3D bioprinted in vitro breast tumor models. The initial mechanical characteristics of each composite hydrogel are correlated to cell proliferation rates and cell spheroid morphology spanning month long culture conditions. MDA-MB-231 breast cancer cells show gel formulation-dependency on the rates and frequency of self-assembly into multicellular tumor spheroids (MCTS). Hydrogel compositions comprised of decreasing alginate concentrations, and increasing gelatin concentrations, result in gels that are mechanically soft and contain a greater number of cell-adhesion moieties driving the development of large MCTS; conversely gels containing increasing alginate, and decreasing gelatin concentrations are mechanically stiffer, with fewer cell-adhesion moieties present in the composite gels yielding smaller and less viable MCTS. These composite hydrogels can be used in the biofabrication of tunable in vitro systems that mimic both the mechanical and biochemical properties of the native tumor stroma.

Directing the Self-assembly of Tumour Spheroids by Bioprinting Cellular Heterogeneous Models within Alginate/Gelatin Hydrogels.

Human tumour progression is a dynamic process involving diverse biological and biochemical events such as genetic mutation and selection in addition to physical, chemical, and mechanical events occurring between cells and the tumour microenvironment. Using 3D bioprinting we have developed a method to embed MDA-MB-231 triple negative breast cancer cells, and IMR-90 fibroblast cells, within a cross-linked alginate/gelatin matrix at specific initial locations relative to each other. After 7 days of co-culture the MDA-MB-231 cells begin to form multicellular tumour spheroids (MCTS) that increase in size and frequency over time. After ~15 days the IMR-90 stromal fibroblast cells migrate through a non-cellularized region of the hydrogel matrix and infiltrate the MDA-MB-231 spheroids creating mixed MDA-MB-231/IMR-90 MCTS. This study provides a proof-of-concept that biomimetic in vitro tissue co-culture models bioprinted with both breast cancer cells and fibroblasts will result in MCTS that can be maintained for durations of several weeks.

[Angioedema as initial manifestation of hypogammaglobulinemia]. / Angioedema como única manifestación inicial de hipogammaglobulinemia.

Common variable immunodeficiency is characterized by hypogammaglobulinemia and the inability to respond to vaccines. Patients mostly manifest infections, however only less than 5 % have pathological conditions as autoimmunity, granulomatous inflammation, and splenomegaly or lymphoproliferative disease among others, without showing infections. We report the case of a woman who debuted with localized cutaneous affection, facial angioedema, without other early symptoms. After diagnosis splenomegaly and bronchiectasis were documented. Angioedema and bronchiectasis responded with IVIG replacement. We also review the dermatological manifestations associated with CVID.

La inmunodeficiencia común variable se caracteriza por hipogammaglobulinemia e incapacidad para responder a las vacunas. Los pacientes en su mayoría manifiestan infecciones, sin embargo, menos de 5 % solo presenta patologías como autoinmunidad, inflamación granulomatosa, esplenomegalia o enfermedad linfoproliferativa, entre otras, sin infecciones. Se describe el caso de una mujer que debutó con afección cutánea localizada, angioedema facial, sin otros síntomas iniciales. Después del diagnóstico se documentó esplenomegalia y bronquiectasias. El angioedema y las bronquiectasias respondieron con la sustitución de la gammaglobulina intravenosa. En este artículo, además de la descripción del caso, se revisan las manifestaciones dermatológicas asociadas con inmunodeficiencia común variable.

Effect of graphene oxide on bacteria and peripheral blood mononuclear cells.

BACKGROUND: Driven by the potential biological applications of graphene, many groups have studied the response of cells exposed to graphene oxide (GO). In particular, investigations of bacteria indicate that there are 2 crucial parameters, which so far have only been investigated separately: GO size and exposure methodology. Our study took into account both parameters. We carefully characterized the samples to catalog sizes and structural properties, and tested different exposure methodologies: exposure in saline solution and in the presence of growth media. Furthermore, we performed experiments with peripheral blood mononuclear cells exposed to our GO materials. METHODS: Atomic force microscopy, scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and transmission electron microscopy were used to characterize the morphology and composition of different samples of GO: GO-H2O, GO-PBS and GO-MG. Our samples had 2D sizes of ~100 nm (GO-H2O and GO-PBS) and >2 µm (GO-MG). We tested antibacterial activity and cytotoxicity toward peripheral blood mononuclear cells of 3 different GO samples. RESULTS: A size-dependent growth inhibition of Escherichia coli (DH5 α) in suspension was found, which proved that this effect depends strongly on the protocol followed for exposure. Hemocompatibility was confirmed by exposing peripheral blood mononuclear cells to materials for 24 hours; viability and apoptosis tests were also carried out. CONCLUSIONS: Our experiments provide vital information for future applications of GO in suspension. If its antibacterial properties are to be potentiated, care should be taken to select 2D sizes in the micrometer range, and exposure should not be carried out in the presence of grow media.

Serum resistin levels are associated with adiposity and insulin sensitivity in obese Hispanic subjects.

BACKGROUND AND AIMS: Resistin is involved in the development of obesity and insulin resistance (IR) in mice and may play a similar role in humans through mechanisms that remain unresolved. The objective of this study was to characterize the relationship between resistin levels in obese subjects with and without IR among Hispanic subjects. MATERIAL AND METHODS: A cross-sectional study was performed on 117 nondiabetic Hispanic subjects of both genders that were allocated into three study groups: A control group (n=47) of otherwise healthy individuals in metabolic balance, a group with obesity (OB) (n=36), and a group with obesity and IR (OB-IR) (n=34). Anthropometric and clinical characterization was carried out, and resistin levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that resistin levels were higher in OB and OB-IR groups when compared to the control group (1331.79±142.15 pg/mL, 1266.28±165.97 pg/mL vs. 959.21±171.43 pg/mL; P<0.05), an effect that was not confounded by age (control, 34.04±10.00 years; OB, 37.30±10.78 years; and OB-IR, 35.67±10.15 years). In addition, we observed a significant correlation (P<0.001) between resistin levels and higher adiposity and insulin sensitivity (IS) in our cohort. CONCLUSIONS: Our results suggest that higher resistin levels are associated with higher adiposity and lower IS among obese Hispanic subjects.

[Guía latinoamericana para el diagnóstico y tratamiento de alergia a las proteínas de la leche de vaca (GL-APLV)].

Cow's milk allergy (CMA) is an immune-based disease that has become an increasing problem. The diagnosis and management of CMA varies from one clinical setting to another and represents a challenge in pediatric practice. In addition, because nonallergic food reactions can be confused with CMA symptoms, there is an overdiagnosis of the disease. In response to these situations, pediatric specialties from recognized institutions throughout Latin America decided to develop a clinical guideline for diagnosis and management of cow's milk allergy. These guidelines include definitions, epidemiology, pathophysiology overview, clinical and evidencebased recommendations for the diagnosis and treatment of CMA. They also include prevention and prognosis sections and identify gaps in the current knowledge to be addressed through future research.

A detailed look at the reaction mechanisms of substituted carbenes with water.

Two competitive reaction mechanisms for the gas-phase chemical transformation of singlet chlorocarbene into chloromethanol in the presence of one and two water molecules are examined in detail. An analysis of bond orders and bond order derivatives as well as of properties of bond critical points in the electron densities along the intrinsic reaction coordinates (IRCs for intermediates → transition state (TS) → products) suggests that, from the perspective of bond breaking/formation, both reactions should be considered to be highly nonsynchronous, concerted processes. Both transition states are early, resembling the intermediates, yielding rate constants whose magnitudes are mostly influenced by structural changes and to a lesser degree by bond breaking/formation. For the case of one water molecule, most of the energy in the reactants region of the IRC is used for structural changes, while the transition state region encompasses the majority of electron activity, except for the formation of the C-O bond, which extends well into the products region. In the case of two water molecules, very little electron flux and comparatively less work required for structural changes is noticed in the reactants region, leading to an earlier transition state and therefore to a smaller activation energy and to a larger rate constant. This, together with evidence gathered from other sources, allows us to provide plausible explanations for the observed difference in rate constants.