Design and Evaluation of PROTACs Targeting Acyl Protein Thioesterase 1.

PROTAC linker design remains mostly an empirical task. We employed the PRosettaC computational software in the design of sulfonyl-fluoride-based PROTACs targeting acyl protein thioesterase 1 (APT1). The software efficiently generated ternary complex models from empirically-designed PROTACs and suggested alkyl linkers to be the preferred type of linker to target APT1. Western blotting analysis revealed efficient degradation of APT1 and activity-based protein profiling showed remarkable selectivity of an alkyl linker-based PROTAC amongst serine hydrolases. Collectively, our data suggests that combining PRosettaC and chemoproteomics can effectively assist in triaging PROTACs for synthesis and providing early data on their potency and selectivity.

Cervical Squamous Cell Carcinoma Diagnosis by FTIR Microspectroscopy.

Cervical cancer was considered the fourth most common cancer worldwide in 2020. In order to reduce mortality, an early diagnosis of the tumor is required. Currently, this type of cancer occurs mostly in developing countries due to the lack of vaccination and screening against the Human Papillomavirus. Thus, there is an urgent clinical need for new methods aiming at a reliable screening and an early diagnosis of precancerous and cancerous cervical lesions. Vibrational spectroscopy has provided very good results regarding the diagnosis of various tumors, particularly using Fourier transform infrared microspectroscopy, which has proved to be a promising complement to the currently used histopathological methods of cancer diagnosis. This spectroscopic technique was applied to the analysis of cryopreserved human cervical tissue samples, both squamous cell carcinoma (SCC) and non-cancer samples. A dedicated Support Vector Machine classification model was constructed in order to categorize the samples into either normal or malignant and was subsequently validated by cross-validation, with an accuracy higher than 90%.

In-Situ Anaerobic Heating of Human Bones Probed by Neutron Diffraction.

The first neutron diffraction study of in-situ anaerobic burning of human bones is reported, aiming at an interpretation of heat-induced changes in bone, which were previously detected by vibrational spectroscopy, including inelastic neutron scattering techniques. Structural and crystallinity variations were monitored in samples of the human femur and tibia, as well as a reference hydroxyapatite, upon heating under anaerobic conditions. Information on the structural reorganization of the bone matrix as a function of temperature, from room temperature to 1000 °C, was achieved. Noticeable crystallographic and domain size variations, together with O-H bond lengths and background variations, were detected. Above 700 °C, the inorganic bone matrix became highly symmetric, devoid of carbonates and organic constituents, while for the lower temperature range (<700 °C), a considerably lower crystallinity was observed. The present pilot study is expected to contribute to a better understanding of the heat-prompted changes in bone, which can be taken as biomarkers of the burning temperature. This information is paramount for bone analysis in forensic science as well as in archeology and may also have useful applications in other biomaterial studies.

4-Oxo-ß-Lactams as Novel Inhibitors for Rhomboid Proteases.

Intramembrane serine proteases (rhomboid proteases) are involved in a variety of biological processes and are implicated in several diseases. Here, we report 4-oxo-ß-lactams as a novel scaffold for inhibition of rhomboids. We show that they covalently react with the active site and that the covalent bond is sufficiently stable for detection of the covalent rhomboid-lactam complex. 4-Oxo-ß-lactams may therefore find future use as both inhibitors and activity-based probes for rhomboid proteases.

Evaluation of the Cytotoxic Effect of Pd2Spm against Prostate Cancer through Vibrational Microspectroscopies.

Regarding the development of new antineoplastic agents, with a view to assess the selective antitumoral potential which aims at causing irreversible damage to cancer cells while preserving the integrity of their healthy counterparts, it is essential to evaluate the cytotoxic effects in both healthy and malignant human cell lines. In this study, a complex with two Pd(II) centers linked by the biogenic polyamine spermine (Pd2Spm) was tested on healthy (PNT-2) and cancer (LNCaP and PC-3) prostate human cell lines, using cisplatin as a reference. To understand the mechanisms of action of both cisplatin and Pd2Spm at a molecular level, Fourier Transform Infrared (FTIR) and Raman microspectroscopies were used. Principal component analysis was applied to the vibrational data, revealing the major metabolic changes caused by each drug, which were found to rely on DNA, lipids, and proteins, acting as biomarkers of drug impact. The main changes were observed between the B-DNA native conformation and either Z-DNA or A-DNA, with a higher effect on lipids having been detected in the presence of cisplatin as compared to Pd2Spm. In turn, the Pd-agent showed a more significant impact on proteins.

A Non-Conventional Platinum Drug against a Non-Small Cell Lung Cancer Line.

A dinuclear Pt(II) complex with putrescine as bridging polyamine ligand ([Pt2Put2(NH3)4]Cl4) was synthesized and assessed as to its potential anticancer activity against a human non-small cell lung cancer line (A549), as well as towards non-cancer cells (BEAS-2B). This effect was evaluated through in vitro cytotoxicity assays (MTT and SRB) coupled to microFTIR and microRaman spectroscopies, the former delivering information on growth-inhibiting and cytotoxic abilities while the latter provided very specific information on the metabolic impact of the metal agent (at the sub-cellular level). Regarding cancer cells, a major impact of [Pt2Put2(NH3)4]Cl4 was evidenced on cellular proteins and lipids, as compared to DNA, particularly via the Amide I and Amide II signals. The effect of the chelate on non-malignant cells was lower than on malignant ones, evidencing a promising low toxicity towards healthy cells.

Breast cancer or surrounding normal tissue? A successful discrimination by FTIR or Raman microspectroscopy.

Breast cancer is a type of cancer with the highest incidence worldwide in 2021, with early diagnosis and rapid treatment intervention being the reasons for the decreasing mortality rate associated with the disease. The major challenge faced by clinicians and pathologists is the lack of accuracy in the histopathological analysis of biopsy or resection samples, leading to classification misdiagnosis and compromising the prognosis of patients. Spectral histopathology has provided great advances regarding cancer diagnosis, especially through the use of FTIR spectroscopy, proving to be a valuable complement to histopathological analyses. In this study unstained formalin-fixed paraffin embedded breast cancer tissue samples, collected from patients undergoing surgery and mounted on glass slides, were probed through FTIR and Raman microspectrocopy. Two classification models were constructed using the AdaBoost algorithm, both achieving >90% accuracy and successfully discriminating invasive breast carcinoma from surrounding normal tissue. Chemical maps from the interfaces of invasive breast carcinoma-surrounding normal tissue were also generated. This study showed the potential of spectral histopathology, in particular FTIR, for daily use in pathology laboratories, introducing few disruptions to the routine workflow while increasing the sensitivity, specificity and accuracy of the diagnoses.

Valorisation Potential of Invasive Acacia dealbata, A. longifolia and A. melanoxylon from Land Clearings.

Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild alkali pretreatments. For example, in A. dealbata stems treated with alkali and P. ostreatus, saccharification yield was 702.3 nmol mg-1, which is higher than the samples treated only with alkali (608.1 nmol mg-1), and 2.9-fold higher than the non-pretreated controls (243.9 nmol mg-1). By characterising biomass and pretreatments, generated data creates value for unused biomass resources, contributing to the implementation of sustainable biorefining systems. In due course, the generated value will lead to economic incentives for landowners to cut back invasive Acacia spp. more frequently, thus reducing excess biomass, which exacerbates wildfire risk.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

The association of osteochemometrics and bone mineral density in humans.

OBJECTIVES: Even though much is known about bone mineral and matrix composition, studies about their relationship with several bone properties and its alterations related to bone diseases such as osteoporosis are practically non-existent in humans. Thus, the development of methods to understand the effects of bone properties at a microscopic level is paramount. This research aimed to evaluate whether Fourier transform infrared-attenuated total reflectance (FTIR-ATR) band intensity ratios correlate with femoral bone mass, bone mineral content (BMC) (total and femoral neck), bone mineral per unit area (BMD) (total, femoral neck, greater trochanter, intertrochanteric region, and Ward's area) and the area (total and femoral neck). A sample of femora from the 21st Century Identified Skeleton Collection (N = 78, 42 females and 36 males) was employed and BMC, BMD, and the femoral areas were acquired by DXA. RESULTS: It was found that only females' BMD had a significant association with the femoral FTIR-ATR indices under study, whereas bone collagen (Am/P) and the content of carbonate Type A (API) in males correlated with the total proximal femur area of the regions of interest and the femoral neck area. DISCUSSION: Men and women showed different changes related to their chemical composition in BMD, BMC, and probed area, most likely due to differences in structure and physiology, as well as mechanical strength in the proximal femoral sites where BMD was analyzed.

Surface Enhanced Raman Spectroscopy for Quantitative Analysis: Results of a Large-Scale European Multi-Instrument Interlaboratory Study.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.

Beyond metrics and morphology: the potential of FTIR-ATR and chemometrics to estimate age-at-death in human bone.

In forensic anthropology, the application of traditional methods for estimating the biological profile of human skeletal remains is often hampered by poor preservation and skeletal representativeness, compromising their reliability. Thus, the development of alternative methods to the morphometric analysis of bones to estimate the biological profile of human remains is paramount. The age of an individual can cause changes in bone morphology, mass and size, as well as in its chemical composition. In this sense, the main objective of this research was to evaluate if the contents of bone collagen (Am/P), carbonate type A (API), carbonate type B (BPI), the relation between the carbonate content (types A and B) to type B carbonate (C/C), carbonate-phosphate ratio (C/P) and crystallinity index (CI), spectroscopic indices obtained from relationships between infrared absorption band intensities (FTIR-ATR), can be used as age-at-death predictors. A sample of femora and humeri from the 21st Century Identified Skeleton Collection (N = 80, 44 females and 36 males) was employed. Results show that, with advancing age, women's femora have lower CI values, but BPI and C/P indices increase, and the deformation and disorder of the crystal lattice are probably affected by the integration of type B carbonate content of the femur. The ratios analysed, especially the CI and the BPI, show potential to estimate age-at-death in human skeletal remains, when sex is already known, thus helping to assess the biological profile when conventional methods cannot be applied.

Chemosteometric regression models of heat exposed human bones to determine their pre-burnt metric dimensions.

OBJECTIVES: Heat exposure can lead to apparently random osteometric changes that hinder the application of metric methods used for biological profiling. The impracticality of using objective and burn-specific osteometric methods reduces the chances of establishing the biological profiles of unknown individuals based on their skeletal remains. We investigated the potential of chemometry analysis based on infrared spectroscopy to predict the amount of heat-induced osteometric changes and how this reflected into sex estimation. MATERIAL AND METHODS: Bones from 41 identified adult skeletons (24 females and 17 males with ages between 62 and 90 years old) were experimentally burnt to maximum temperatures ranging from 450°C to 1,100°C (attained after 65 to 240 min). Measurements were taken both before and after each experiment and powder samples were analyzed through FTIR-ATR. Correlations among heat-induced metric changes and chemometric indices (crystallinity index; B-type carbonates; carbonate [A + B] to carbonate B ratio; hydroxyl to phosphate ratio; 630 cm-1 , 1450 cm-1 , 3572 cm-1 , and 3642 cm-1 ) were tested. Significant variables were used to build regression models to predict heat-induced metric change which were then tested on an independent set of samples. Agreement in sex estimation between the pre- and post-burnt samples was also evaluated. RESULTS: All indices were significantly correlated to heat-induced metric changes (α = .01) and the highest correlations were obtained for the 630 cm-1 , 3572 cm-1 , and crystallinity index. We confirmed that regression models based on chemometrics obtained from infrared spectra through FTIR-ATR are better at estimating heat-induced metric changes affecting bone and at sexing remains than other osteometric methods such as those based on correction factors or on metric references specific to calcined bones. DISCUSSION: Regression models avoid the subjectivity associated with the application of other methods. While the latter can be applied only to calcined bones, which is difficult to assess sometimes, regression models can be applied to all bones regardless of their condition. Also, regression models have the advantage of allowing to infer about heat-induced metric change on a case-by-case basis.

Phylogenetic analysis of Hepatozoon spp. (Apicomplexa: Hepatozoidae) infecting Philodryas patagoniensis (Serpentes: Dipsadidae) in Uruguay.

The purpose of this study was to perform a phylogenetic analysis of Hepatozoon spp. infecting Philodryas patagoniensis in Uruguay. Twenty-five road-killed specimens of P. patagoniensis from ten departments were obtained. Samples of blood and/or heart tissue were taken. Polymerase chain reaction (PCR) assay was carried out amplifying a specific target region of the 18S rRNA gene of Hepatozoon spp. Eighteen out of twenty-five samples were positive to Hepatozoon spp., which gave an overall prevalence of 72%. Phylogenetic analyses with the obtained sequences were carried out to determine the relationship with closely related species found in the region. The results revealed that samples were split into two clades with a high bootstrap support. Clade I was formed with Hepatozoon spp. sequences obtained in this study from P. patagoniensis, Hepatozoon cuestensis from Crotalus durissus terrificus and Hepatozoon musa from Philodryas nattereri, and Hepatozoon spp. retrieved from Cerdocyon thous, Hemidactylus mabouia, and Phyllopezus pollicaris from Brazil, respectively. Clade II was grouped with Hepatozoon cevapii and Hepatozoon massardii, both species described for C. d. terrificus from Brazil. This is the first report of Hepatozoon spp. in snakes from Uruguay.

A New Look into the Mode of Action of Metal-Based Anticancer Drugs.

The mode of action of Pt- and Pd-based anticancer agents (cisplatin and Pd2Spm) was studied by characterising their impact on DNA. Changes in conformation and mobility at the molecular level in hydrated DNA were analysed by quasi-elastic and inelastic neutron scattering techniques (QENS and INS), coupled to Fourier transform infrared (FTIR) and microRaman spectroscopies. Although INS, FTIR and Raman revealed drug-triggered changes in the phosphate groups and the double helix base pairing, QENS allowed access to the nanosecond motions of the biomolecule's backbone and confined hydration water within the minor groove. Distinct effects were observed for cisplatin and Pd2Spm, the former having a predominant effect on DNA´s spine of hydration, whereas the latter had a higher influence on the backbone dynamics. This is an innovative way of tackling a drug´s mode of action, mediated by the hydration waters within its pharmacological target (DNA).

Borrelia burgdorferi sensu lato infecting Ixodes auritulus ticks in Uruguay.

In the southern cone of South America different haplotypes of Borrelia burgdorferi sensu lato (Bbsl) have been detected in Ixodes spp. from Argentina, southern Brazil, Chile, and Uruguay. So far, Lyme borreliosis has not been diagnosed in Uruguay and the medical relevance of the genus Ixodes in South America is uncertain. However, the growing number of new genospecies of Bbsl in the southern cone region and the scarce information about its pathogenicity, reservoirs and vectors, highlights the importance of further studies about spirochetes present in Uruguay and the region. The aim of this study was to determine the presence of Bbsl in Ixodes auritulus ticks collected from birds and vegetation in two localities of southeastern Uruguay. In total 306 I. auritulus were collected from 392 passerine birds sampled and 1110 ticks were collected by flagging in vegetation. Nymphs and females were analyzed for Borrelia spp. by PCR targeting the flagellin (fla) gene and the rrfA-rrlB intergenic spacer region (IGS). The phylogenetic analysis of Borrelia spp. positive samples from passerine birds and vegetation revealed the presence of four fla haplotypes that form a clade within the Bbsl complex. They were closely related to isolates of Borrelia sp. detected in I. auritulus from Argentina and Canada.

Anticancer drug impact on DNA - a study by neutron spectroscopy coupled with synchrotron-based FTIR and EXAFS.

Complementary structural and dynamical information on drug-DNA interplay has been achieved at a molecular level, for Pt/Pd-drugs, allowing a better understanding of their pharmacodynamic profile which is crucial for the development of improved chemotherapeutic agents. The interaction of two cisplatin-like dinuclear Pt(ii) and Pd(ii) complexes with DNA was studied through a multidisciplinary experimental approach, using quasi-elastic neutron scattering (QENS) techniques coupled with synchrotron-based extended X-ray absorption fine structure (SR-EXAFS) and Fourier-Transform Infrared Spectroscopy-Attenuated Total Reflectance (SR-FTIR-ATR). DNA extracted from drug-exposed human triple negative breast cancer cells (MDA-MB-231) was used, with a view to evaluate the effect of the unconventional antineoplastic agents on this low prognosis type of cancer. The drug impact on DNA's dynamical profile, via its hydration layer, was provided by QENS, a drug-triggered enhanced mobility having been revealed. Additionally, an onset of anharmonicity was detected for dehydrated DNA, at room temperature. Far- and mid-infrared measurements allowed the first simultaneous detection of the drugs and their primary pharmacological target, as well as the drug-prompted changes in DNA's conformation that mediate cytotoxicity. The local environment of the absorbing Pd(ii) and Pt(ii) centers in the drugs' adducts with adenine, guanine and glutathione was attained by EXAFS.

FTIR Spectroscopy and DFT Calculations to Probe the Kinetics of ß-Carotene Thermal Degradation.

The thermal degradation of ß-carotene in air was investigated. The sample was heated at different temperatures (90, 100, 115, and 130 °C) for periods of up to 8 h to perform a complete kinetic study, the product analysis having been carried out via infrared spectroscopy in attenuated total reflectance mode coupled to density functional theory (DFT) calculations. The kinetics of this thermal degradation process was found to follow a first-order scheme, with rate coefficients varying from k90 °C = (2.0 ± 0.3) × 10-3 to k130 °C = (11.0 ± 0.7) × 10-3 min-1, the experimental activation energy having been calculated as (52 ± 1) kJ mol-1. This Ea value is close to the DFT energies corresponding to a C15-15' or a C13-14 cis-trans isomerization, followed by the formation of a carotene-oxygen diradical, which was characterized for the first time. Comparison between the experimental and calculated infrared data confirmed the C15-15'- cis rupture as the predominant reaction pathway and retinal as the major degradation product.

Potential of Bioapatite Hydroxyls for Research on Archeological Burned Bone.

The estimation of the maximum temperature affecting skeletal remains was previously attempted via infrared techniques. However, fossilization may cause changes in the composition of bones that replicate those from burned bones. We presently investigated the potential of three OH/P indices (intensity ratios of characteristic infrared bands for OH and phosphate groups, respectively) to identify bones burned at high temperatures (>800 °C) and to discriminate between fossil and burned archeological bones, using vibrational spectroscopy: combined inelastic neutron scattering (INS) and FTIR-ATR. The INS analyses were performed on two unburned samples and 14 burned samples of human femur and humerus. FTIR-ATR focused on three different samples: (i) modern bones comprising 638 unburned and 623 experimentally burned (400-1000 °C) samples; (ii) archeological cremated human skeletal remains from the Bronze and Iron Ages comprising 25 samples; and (iii) fossil remains of the Reptilia class from the Middle Triassic to the Eocene. The OH/P indices investigated were 630 cm-1/603 cm-1, 3572 cm-1/603 cm-1, and 3572 cm-1/1035 cm-1. The OH signals became visible in the spectra of recent and archeological bones burned between 600 and 700 °C. Although they have episodically been reported in previous works, no such peaks were observed in our fossil samples thus suggesting that this may be a somewhat rare event. While high crystallinity index values should always correspond to clearly visible hydroxyl signals in burned bone samples, this is not always the case in fossils which may be used as a criterion to exclude burning as the agent responsible for high crystallinity ratios.