HIV-1 early and late diagnosis in the Emilia Romagna Region (Italy): a three year study.

It is crucial to establish the timing of infection and distinguish between early and long-lasting HIV-1 infections not only for partner notification and epidemiological surveillance, but also to offer early drug treatment and contain the spread of infection. This study analyzed serum and/or plasma samples with a first positive HIV antibody/antigen result coming from different Medical Centers in the Emilia Romagna Region, North East Italy, using the avidity assay, Western Blotting, RNA viral load, CD4 cell counts and genotyping assay. From May 2013 to May 2016, we certified 845 new HIV-1 infections, 18.7% of which were classified on the basis of avidity index as recent infections and 81.3% as long-lasting infections, with an estimated conversion time exceeding six months at the time of study. Western Blotting showed reactivity to only one or two HIV-1 proteins in recently infected patients (RIPs), while a complete pattern to gag, env and pol proteins was observed in most long-lasting infected patients (LLIPs). The median age, gender, nationality and risk transmission factors were comparable in RIPs and LLIPs. Phylogenetic analysis performed in available plasma disclosed B strains, non-B subtypes and circulating recombinant forms (CRFs) in both groups of patients, with a major presence of CRFs in non-Italian HIV subjects. The large number of patients unaware of their HIV status makes it crucial to discover hidden epidemics and implement appropriate targeted public health interventions.

Helicobacter pylori and the Human Gastrointestinal Microbiota: A Multifaceted Relationship.

Helicobacter pylori is a type of Gram-negative bacteria belonging to the Proteobacteria phylum which is known to cause gastrointestinal disorders such as gastritis and gastric ulcers. Its treatment is based on current eradication regimens, which are composed of combinations of antibiotics such as clarithromycin, metronidazole, levofloxacin and amoxicillin, often combined with a proton pump inhibitor (PPI). With the development of sequencing technologies, it has been demonstrated that not only does the colonization of the gastric and gut environment by H. pylori cause microbial changes, but also the treatment regimens used for its eradication have a significant altering effect on both the gastric and gut microbiota. Here, we review current knowledge on microbiota modulations of current therapies in both environments. We also summarize future perspectives regarding H. pylori infection, the integration of probiotics into therapy and what challenges are being faced on a global basis when we talk about eradication.

Kilometer-range depth imaging at 1,550 nm wavelength using an InGaAs/InP single-photon avalanche diode detector.

We have used an InGaAs/InP single-photon avalanche diode detector module in conjunction with a time-of-flight depth imager operating at a wavelength of 1550 nm, to acquire centimeter resolution depth images of low signature objects at stand-off distances of up to one kilometer. The scenes of interest were scanned by the transceiver system using pulsed laser illumination with an average optical power of less than 600 µW and per-pixel acquisition times of between 0.5 ms and 20 ms. The fiber-pigtailed InGaAs/InP detector was Peltier-cooled and operated at a temperature of 230 K. This detector was used in electrically gated mode with a single-photon detection efficiency of about 26% at a dark count rate of 16 kilocounts per second. The system's overall instrumental temporal response was 144 ps full width at half maximum. Measurements made in daylight on a number of target types at ranges of 325 m, 910 m, and 4.5 km are presented, along with an analysis of the depth resolution achieved.

Osteobiography of a 19th century elderly woman with pertrochanteric fracture and osteoporosis: a multidisciplinary approach.

In this paper the osteobiography of an elderly woman recovered from a cemetery tomb where she was buried in 1850, affected by hip fracture and osteoporosis, is described. The overall anthropological characteristics of the individual have been investigated. Macroscopic, radiographic, tomographic, microscopic, and chemical and structural examinations have been performed to give a detailed account of the condition of the skeleton. A non-union pertrochanteric fracture not surgically treated and probably due to senile osteoporosis was diagnosed. The consequences of the fracture to the bones show that this individual likely survived several years following the injury. The osseous features we describe (remodelled bone at the fracture site, asymmetry of entheseal changes likely related to the particular walking pattern of the individual) may be useful in personal identification of skeletons of legal interest. Regarding the recognition of osteoporosis in unearthed skeletons, our study underlines that the cortical thickness, microscopic features, degree of crystallinity and Ca/P ratio represent more useful elements than the mean bone density, mineral/matrix ratio and mineral maturity, which are more sensitive to diagenetic changes that affect the mineral phase post-mortem.

A quantum-enhanced wide-field phase imager.

Quantum techniques can be used to enhance the signal-to-noise ratio in optical imaging. Leveraging the latest advances in single-photon avalanche diode array cameras and multiphoton detection techniques, here, we introduce a supersensitive phase imager, which uses space-polarization hyperentanglement to operate over a large field of view without the need of scanning operation. We show quantum-enhanced imaging of birefringent and nonbirefringent phase samples over large areas, with sensitivity improvements over equivalent classical measurements carried out with equal number of photons. The potential applicability is demonstrated by imaging a biomedical protein microarray sample. Our technology is inherently scalable to high-resolution images and represents an essential step toward practical quantum-enhanced imaging.

Tendon and ligament fibrillar crimps give rise to left-handed helices of collagen fibrils in both planar and helical crimps.

Collagen fibres in tendons and ligaments run straight but in some regions they show crimps which disappear or appear more flattened during the initial elongation of tissues. Each crimp is formed of collagen fibrils showing knots or fibrillar crimps at the crimp top angle. The present study analyzes by polarized light microscopy, scanning electron microscopy, transmission electron microscopy the 3D morphology of fibrillar crimp in tendons and ligaments of rat demonstrating that each fibril in the fibrillar region always twists leftwards changing the plane of running and sharply bends modifying the course on a new plane. The morphology of fibrillar crimp in stretched tendons fulfills the mechanical role of the fibrillar crimp acting as a particular knot/biological hinge in absorbing tension forces during fibril strengthening and recoiling collagen fibres when stretching is removed. The left-handed path of fibrils in the fibrillar crimp region gives rise to left-handed fibril helices observed both in isolated fibrils and sections of different tendons and ligaments (flexor digitorum profundus muscle tendon, Achilles tendon, tail tendon, patellar ligament and medial collateral ligament of the knee). The left-handed path of fibrils represents a new final suprafibrillar level of the alternating handedness which was previously described only from the molecular to the microfibrillar level. When the width of the twisting angle in the fibrillar crimp is nearly 180 degrees the fibrils appear as left-handed flattened helices forming crimped collagen fibres previously described as planar crimps. When fibrils twist with different subsequent rotational angles (< 180 degrees ) they always assume a left-helical course but, running in many different nonplanar planes, they form wider helical crimped fibres.

Collagen fibre arrangement and functional crimping pattern of the medial collateral ligament in the rat knee.

Ligaments have been described as multifascicular structures with collagen fibres cross-connecting to each other or running straight and parallel also showing a waviness or crimping pattern playing as a shock absorber/recoiling system during joint motions. A particular collagen array and crimping pattern in different ligaments may reflect different biomechanical roles and properties. The aim of the study was to relate the 3D collagen arrangement in the crimping pattern of the medial collateral ligament (MCL) to its functional role. The MCL is one of the most injured ligaments during sports activities and an experimental model to understand the rate, quality and composition of ligaments healing. A deep knowledge of structure-function relationship of collagen fibres array will improve the development of rehabilitation protocols and more appropriate exercises for recovery of functional activity. The rat MCL was analysed by polarized light microscopy, confocal laser microscopy and scanning electron microscopy (SEM). Histomorphometric analysis demonstrated that MCL crimps have a smaller base length versus other tendons. SEM observations demonstrated that collagen fibres showing few crimps were composed of fibrils intertwining and crossing one another in the outer region. Confocal laser analyses excluded a helical array of collagen fibres. By contrast, in the core portion, densely packed straight collagen fibres ran parallel to the main axis of the ligament being interrupted both by planar crimps, similar to tendon crimps, and by newly described right-handed twisted crimps. It is concluded that planar crimps could oppose or respond exclusively to tensional forces parallel to the main ligament axis, whereas the right-handed twisted crimps could better resist/respond to a complex of tensional/rotational forces within the ligament thus opposing to an external rotation of tibia.

Correlative microscopy of bone in implant osteointegration studies.

Routine morphological analyses usually include investigations by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Each of these techniques provides specific information on tissue morphology and all the obtained results are then combined to give an in-depth morphological overview of the examined sample. The limitations of this traditional comparative microscopy lie in the fact that each technique requires a different experimental sample, so that many specimens are necessary and the combined results come from different samples. The present study describes a technical procedure of correlative microscopy, which allows us to examine the same bone section first by LM and then, after appropriate processing, by SEM or TEM. Thanks to the possibility of analyzing the same undecalcified bone sections both by LM and SEM, the approach described in the present study allows us to make very accurate evaluations of old/new bone morphology at the bone-implant interface.

Structure relates to elastic recoil and functional role in quadriceps tendon and patellar ligament.

Tendons and ligaments have similar but slightly different structure and composition. Crimps of tendons and ligaments are morphological structures related to the elastic functional properties of these connective tissues. Aim of this study was to investigate the morphological arrangement of collagen fibres, fibrils and crimping pattern of suprapatellar (rectus femoris tendon-RFT and vastus intermedius tendon-VIT) and infrapatellar connective tissues (patellar ligament-PL) to relate their structural aspects to their common function role of leg extension. RFT, VIT and PL were removed from knees of Sprague-Dawley rats and light and electron microscopy (TEM and SEM) performed. Sagittal sections showed that collagen array and crimping pattern were similar in RFT and PL but differed from VIT. Morphometric analysis confirmed that crimp number was about the same in RFT and PL (5.4+/-1.4 and 6.1+/-2.8 respectively), but it was almost three times higher in VIT (14.5+/-4.7). Similarly crimp top angle in RFT and PL (141.5+/-15.0 degrees and 146.2+/-12.2 degrees respectively) was significantly higher than in VIT (122.3+/-14.8 degrees ) and the crimp base length was more than twice as wide in RFT (75.5+/-22.6microm) and PL (72.3+/-28.9microm) than in VIT (36+/-14.1microm). The smaller, fewer and most crimped crimps in VIT show that this tendon has a greater elastic recoil and responds to higher forces as among quadriceps muscles the vastus intermedius belly contributes the most during knee extension. By contrast, RFT acting as a "stopper" tendon also plays a ligament role by limiting an excessive flexion of the joint during postural rest position of the knee.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BACKGROUND: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. METHODS: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. RESULTS: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. CONCLUSION: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

Different crimp patterns in collagen fibrils relate to the subfibrillar arrangement.

Collagen fibril ultrastructure and course were examined in different connective tissues by PLM, SEM, TEM, and AFM. In tendons, collagen fibrils were large and heterogeneous with a straight subfibrillar arrangement. They ran densely packed, parallel, and straight changing their direction only in periodic crimps where fibrils showed a local deformation (fibrillar crimps). Other tissues such as aponeurosis, fascia communis, skin, aortic wall, and tendon and nerve sheaths showed thinner uniform fibrils with a helical subfibrillar arrangement. These fibrils appeared in parallel or helical arrangement following a wavy, undulating course. Ligaments showed large fibrils as in tendon, with fibrillar crimps but less packed. Thinner uniform-sized fibrils also were observed. Fibrillar crimps seem to be related to the subfibrillar arrangement being present only in large fibrils with a straight subfibrillar arrangement. These stiffer fibrils respond mainly to unidirectional tensional forces, whereas the flexible thinner fibrils with helical subfibrils can accommodate extreme curvatures without harm, thus responding to multidirectional loadings.

Decorin transfection induces proteomic and phenotypic modulation in breast cancer cells 8701-BC.

Decorin is a prototype member of the small leucine-rich proteoglycan family widely distributed in the extracellular matrices of many connective tissues, where it has been shown to play multiple important roles in the matrix assembly process, as well as in some cellular activities. A major interest for decorin function concerns its role in tumorigenesis, as growth-inhibitor of different neoplastic cells, and potential antimetastatic agent. The aim of our research was to investigate wide-ranged effects of transgenic decorin on breast cancer cells. To this purpose we utilized the well-characterized 8701-BC cell line, isolated from a ductal infiltrating carcinoma of the breast, and two derived decorin-transfected clones, respectively, synthesizing full decorin proteoglycan or its protein core. The responses to the ectopic decorin production were examined by studying morphological changes, cell proliferation rates, and proteome modulation. The results revealed new important antioncogenic potentialities, likely exerted by decorin through a variety of distinct biochemical pathways. Major effects included the downregulation of several potential breast cancer biomarkers, the reduction of membrane ruffling, and the increase of cell-cell adhesiveness. These results disclose original aspects related to the reversion of malignant traits of a prototype of breast cancer cells induced by decorin. They also raise additional interest for the postulated clinical application of decorin.

Influence of different implant surfaces on peri-implant osteogenesis: histomorphometric analysis in sheep.

BACKGROUND: The present study investigated peri-implant osteogenesis and implant biologic fixation in different zirconia sandblasted endosseous titanium surfaces (SLA-60 and SLA-120) and a turned titanium surface (T) 2 and 4 weeks after surgery. METHODS: Seventy-two implant screws were implanted in tibia of six sheep. Histologic sections of implants (2 and 4 weeks after surgery) were analyzed with light microscopy for histomorphometric analysis of bone-to-implant contact (BIC), bone ingrowth (BI), and bone surface (BS/BV). Histologic blocks were used to perform bone microhardness studies next to the implants. Some implants were also observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: In general, the highest values of BIC, BI, BS/BV, and Vickers hardness number (HV) were measured in SLA-60 samples, followed by SLA-120 and T implants. Two weeks after surgery, all the implants appeared biologically fixed by a newly formed woven bone arranged in thin bone trabeculae and filling the gap between implant and host bone. Four weeks after implantation, the thickness of the woven bone trabeculae had increased, especially around the SLA-60 and SLA-120 implants by a gradual deposition of parallel-fiber bone. CONCLUSIONS: Our results suggest that, in the early period of peri-implant healing, the implant surface morphology that seemed to influence the increase of peri-implant osteogenesis, bone turnover, and peri-implant bone maturation was SLA-60. We suggest that this surface, characterized by moderately deep titanium cavities very similar to the osteocyte lacunae, could act as a microscopic scaffold for mesenchymal and/or osteoblast-like cells adhesion.

Diffuse optical characterization of collagen absorption from 500 to 1700 nm.

Reduction in scattering, high absorption, and spectral features of tissue constituents above 1000 nm could help in gaining higher spatial resolution, penetration depth, and specificity for in vivo studies, opening possibilities of near-infrared diffuse optics in tissue diagnosis. We present the characterization of collagen absorption over a broadband range (500 to 1700 nm) and compare it with spectra presented in the literature. Measurements were performed using a time-domain diffuse optical technique. The spectrum was extracted by carefully accounting for various spectral distortion effects, due to sample and system properties. The contribution of several tissue constituents (water, lipid, collagen, oxy, and deoxy-hemoglobin) to the absorption properties of a collagen-rich in vivo bone location, such as radius distal in the 500- to 1700-nm wavelength region, is also discussed, suggesting bone diagnostics as a potential area of interest.

Self-aggregation of fibrillar collagens I and II involves lysine side chains.

Several properties of fibrillar collagens depend on abundance and position of ionic amino acids. We recently demonstrated that N-methylation and N-acetylation of Lys/Hyl amino group did not significantly alter the thermal stability of the triple helical conformation and that the binding of modified collagens I and II to decorin is lost only on N-acetylation. The positive charge at physiological pH of Lys/Hyl side chains is preserved only by N-methylation. We report here the new aspect of the influence of the same modifications on collagen self-aggregation in neutral conditions. Three collagen preparations are very differently affected by N-methylation: acid-soluble type I collagen maintains the ability to form banded fibrils with 67-nm periodicity, whereas almost no structured aggregates were detected for pepsin-soluble type I collagen; pepsin-soluble type II collagen forms a very different supramolecular species, known as segment long spacing (SLS). N-acetylation blocks the formation of banded fibrils in neutral conditions (as did all other chemical modifications reported in the literature), demonstrating that the positive charge of Lys/Hyl amino groups is essential for self-aggregation. Kinetic measurements by turbidimetry showed a sizeable increase of absorbance only for the two N-methylated samples forming specific supramolecular aggregates; however, the derivatization affects aggregation kinetics by increasing lag time and decreasing maximum slope of absorbance variation, and lowers aggregation competency. We discuss that the effects of N-methylation on self-aggregation are caused by fewer or weaker salt bridges and by decrease of hydrogen bonding potential and conclude that protonated Lys side chains are involved in the fibril formation process.

The 3D structure of crimps in the rat Achilles tendon.

The ultrastructure of crimps of the Achilles tendon of rat, excised and processed in a slack condition, was investigated by atomic force microscopy in air, in fluid and by scanning electron microscopy and stereo reconstruction. The tendon was made of distinct fascicles, each comprising a succession of straight segments connected by sharp angles. The length of the segments and the interposed angles varied widely. In particular, the angles ranged from almost zero to over 135 degrees . We did not observe a unique structure for the hinge regions, but rather a variety of gradations of buckling and/or torsion with no evident correlation with other features of tendon. A constant hallmark was the local loss of regular molecular packing, as revealed by the disappearance of the D-banding. Our results do not support recent reports of a helical structure or smooth sinusoidal waves in tendons. Such structures may nonetheless exist in other non-tensile structures whose collagen fibrils exhibit a helical inner architecture and are able to follow a highly convoluted course without buckling or crimping.

Different titanium surface treatment influences human mandibular osteoblast response.

BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.

Osteogenesis and morphology of the peri-implant bone facing dental implants.

This study investigated the influence of different implant surfaces on peri-implant osteogenesis and implant face morphology of peri-implant tissues during the early (2 weeks) and complete healing period (3 months). Thirty endosseous titanium implants (conic screws) with differently treated surfaces (smooth titanium = SS, titanium plasma sprayed = TPS, sand-blasted zirconium oxide = Zr-SLA) were implanted in femur and tibiae diaphyses of two mongrel sheep. Histological sections of the implants and surrounding tissues obtained by sawing and grinding techniques were observed under light microscopy (LM). The peri-implant tissues of other samples were mechanically detached from the corresponding implants to be processed for SEM observation. Two weeks after implantation, we observed osteogenesis (new bone trabeculae) around all implant surfaces only where a gap was present at the host bone-metal interface. No evident bone deposition was detectable where threads of the screws were in direct contact with the compact host bone. Distance osteogenesis predominated in SS implants, while around rough surfaces (TPS and Zr-SLA), both distance and contact osteogenesis were present. At SEM analysis 2 weeks after implantation, the implant face of SS peri-implant tissue showed few, thin, newly formed, bone trabeculae immersed in large, loose, marrow tissue with blood vessels. Around the TPS screws, the implant face of the peri-implant tissue was rather irregular because of the rougher metal surface. Zr-SLA screws showed more numerous, newly formed bone trabeculae crossing marrow spaces and also needle-like crystals in bone nodules indicating an active mineralising process. After 3 months, all the screws appeared osseointegrated, being almost completely covered by a compact, mature, newly formed bone. However, some marrow spaces rich in blood vessels and undifferentiated cells were in contact with the metal surface. By SEM analysis, the implant face of the peri-implant tissue showed different results. Around the SS screws, the compact bone with areas of different mineralisation rate appeared very smooth, while around the rougher TPS screws, the bone still showed an irregular surface corresponding to the implant macro/microroughness. Around the Zr-SLA screws, a more regular implant-bone surface and sparse, calcified marrow spaces were detectable. Results from this research suggest that 2 weeks after implantation, trabecular bone represents the calcified healing tissue, which supports the early biological fixation of the implants. The peri-implant marrow spaces, rich in undifferentiated cells and blood vasculature, observed both 2 weeks and 3 months after surgery, favour the biological turnover of both early and mature peri-implant bone. The implant surface morphology strongly influences the rate and the modality of peri-implant osteogenesis, as do the morphology and arrangement of the implant face in peri-implant bone both during early healing (after 2 weeks) and when the implant is just osseointegrated; rough surfaces, and in particular Zr-SLA, seem to better favour bone deposition on the metal surface.

Time-gated single-photon detection module with 110 ps transition time and up to 80 MHz repetition rate.

We present the design and characterization of a complete single-photon counting module capable of time-gating a silicon single-photon avalanche diode with ON and OFF transition times down to 110 ps, at repetition rates up to 80 MHz. Thanks to this sharp temporal filtering of incoming photons, it is possible to reject undesired strong light pulses preceding (or following) the signal of interest, allowing to increase the dynamic range of optical acquisitions up to 7 decades. A complete experimental characterization of the module highlights its very flat temporal response, with a time resolution of the order of 30 ps. The instrument is fully user-configurable via a PC interface and can be easily integrated in any optical setup, thanks to its small and compact form factor.