Intravital Imaging of a Massive Lymphocyte Response in the Cortical Dura of Mice after Peripheral Infection by Trypanosomes

Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma.     

Structural and Mechanical Improvements to Bone Are Strain Dependent with Axial Compression of the Tibia in Female C57BL/6 Mice

Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function.     

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull^1,2 or thinned-skull ³ preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX₃CR₁ GFP/+ mice ⁴ to visualize microglia with enhanced green fluorescent protein to ≤150 μm beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.     

Altered Mechanical Environment of Bone Cells in an Animal Model of Short- and Long-Term Osteoporosis

Alterations in bone tissue composition during osteoporosis likely disrupt the mechanical environment of bone cells and may thereby initiate a mechanobiological response. It has proved challenging to characterize the mechanical environment of bone cells in vivo, and the mechanical environment of osteoporotic bone cells is not known. The objective of this research is to characterize the local mechanical environment of osteocytes and osteoblasts from healthy and osteoporotic bone in a rat model of osteoporosis. Using a custom-designed micromechanical loading device, we apply strains representative of a range of physical activity (up to 3000 με) to fluorescently stained femur samples from normal and ovariectomized rats. Confocal imaging was simultaneously performed, and digital image correlation techniques were applied to characterize cellular strains. In healthy bone tissue, osteocytes experience higher maximum strains (31,028 ± 4213 με) than osteoblasts (24,921 ± 3,832 με), whereas a larger proportion of the osteoblast experiences strains >10,000 με. Most interestingly, we show that osteoporotic bone cells experience similar or higher maximum strains than healthy bone cells after short durations of estrogen deficiency (5 weeks), and exceeded the osteogenic strain threshold (10,000 με) in a similar or significantly larger proportion of the cell (osteoblast, 12.68% vs. 13.68%; osteocyte, 15.74% vs. 5.37%). However, in long-term estrogen deficiency (34 weeks), there was no significant difference between bone cells in healthy and osteoporotic bone. These results suggest that the mechanical environment of bone cells is altered during early-stage osteoporosis, and that mechanobiological responses act to restore the mechanical environment of the bone tissue after it has been perturbed by ovariectomy.     

Label-free multimodal quantitative imaging flow assay for intrathrombus formation in vitro

Microfluidics in vitro assays recapitulate a blood vessel microenvironment using surface-immobilized agonists under biofluidic flows. However, these assays do not quantify intrathrombus mass and activities of adhesive platelets at the agonist margin and use fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a label-free multimodal quantitative imaging flow assay that combines rotating optical coherent scattering microscopy and quantitative phase microscopy. The combined imaging platform enables real-time evaluation of membrane fluctuations of adhesive-only platelets and total intrathrombus mass under physiological flow rates in vitro. We call this multimodal quantitative imaging flow assay coherent optical scattering and phase interferometry (COSI). COSI records intrathrombus mass to picogram accuracy and shape changes to a platelet membrane with high spatial-temporal resolution (0.4 μm/s) under physiological and pathophysiological fluid shear stress (1800 and 7500 s⁻¹). With COSI, we generate an axial slice of 4 μm from the coverslip surface, approximately equivalent to the thickness of a single platelet, which permits nanoscale quantification of membrane fluctuation (activity) of adhesive platelets during initial adhesion, spreading, and recruitment into a developing thrombus (mass). Under fluid shear, pretreatment with a broad range metalloproteinase inhibitor (250 μM GM6001) blocked shedding of platelet adhesion receptors that shown elevated adhesive platelet activity at average of 42.1 μm/s and minimal change in intrathrombus mass.     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

Cryptocotyle lata (Trematoda: Heterophyidae) Adult from a Korean Raccoon Dog,Nyctereutes procyonoides koreensis

Total 513 heterophyid flukes were collected from a carcass of wild Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. With morphological and molecular characteristics, the flukes were identified to Cryptocotyle lata. The adult C. lata were minute, transparent, pentagonal, 522 μm long by 425 μm wide. Ceca extended into post-testicular region. Ventrogenital sac elliptical, 79 μm by 87 μm with genital pore and ventral sucker. Two testes semielliptical and slightly lobed, located in the posterior region, right testis 173 μm by 155 μm, left testis 130 μm by 134 μm. In a phylogenetic tree, the fluke specimen of this study was grouped with C. lata divergent from Cryptocotyle lingua. We report here N. procyonoides koreensis first as a natural definitive host of C. lata.     

Monitoring Bacterial Burden,Inflammation and Bone Damage Longitudinally Using Optical and μCT Imaging in an Orthopaedic Implant Infection in Mice

Background

Recent advances in non-invasive optical, radiographic and μCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure.

Methodology/Principal Findings

An orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×10³ CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and μCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with μCT images. To quantify the anatomical bone changes on the μCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection.

Conclusions/Significance

Bioluminescent and fluorescent optical imaging was combined with X-ray and μCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.     

Application of Thinned-Skull Cranial Window to Mouse Cerebral Blood Flow Imaging Using Optical Microangiography

In vivo imaging of mouse brain vasculature typically requires applying skull window opening techniques: open-skull cranial window or thinned-skull cranial window. We report non-invasive 3D in vivo cerebral blood flow imaging of C57/BL mouse by the use of ultra-high sensitive optical microangiography (UHS-OMAG) and Doppler optical microangiography (DOMAG) techniques to evaluate two cranial window types based on their procedures and ability to visualize surface pial vessel dynamics. Application of the thinned-skull technique is found to be effective in achieving high quality images for pial vessels for short-term imaging, and has advantages over the open-skull technique in available imaging area, surgical efficiency, and cerebral environment preservation. In summary, thinned-skull cranial window serves as a promising tool in studying hemodynamics in pial microvasculature using OMAG or other OCT blood flow imaging modalities.     

Evaluation of Granulated Lactose as a Carrier for DPI Formulations 1: Effect of Granule Size

The objective of this study was to investigate the effect of large granulated lactose carrier particle systems on aerosol performance of dry powder inhaler formulations. Granulated lactose carriers with average sizes ranging from 200 to 1,000 μm were prepared and subsequently fractionated into separate narrow size powders. The fractionated granulated lactose (GL) samples were characterized in terms of size, specific surface area, surface roughness, morphology, density, flowability, and solid-state. The in vitro aerosolization performance was performed on the different size fractions of GL samples from a commercial inhaler device (Aerolizer®) with a model formulation (2% w/w salbutamol sulfate). The cascade impaction parameters employed were 60 or 90 L/min with standard (aperture size, 0.6 mm) or modified piercing holes (aperture size, 1.2 mm) of the inhaler loaded capsules. It was shown that the largest size fraction formulation (850–1000 μm) had a slight improvement in the fine particle fraction (FPF) compared to immediately preceding size fractions, explained by a smaller adhesive force between drug and carrier. Compared to commercial piercing holes, enlarged piercing holes generated a slight decreasing trend of FPF as the lactose powder sizes increased from 200–250 μm to 600–850 μm, perhaps due to the reduced detachment force by flow forces. The size, surface roughness, density, and flowability of lactose carrier as well as device design all contributed to the aerosol dispersion performance of granulated lactose-based adhesive mixtures. It was concluded that poorer or enhanced redispersion performance is not an inherent property to the significantly large size of granulated lactose carriers as previously contended.KEY WORDS: adhesive force, carrier roughness, carrier size, DPI formulations, granulated lactose     

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 μm circle plasmid (2 μm). Here we show that accumulation of 2 μm in the SUMO pathway mutants siz1Δ siz2Δ, slx5Δ, and slx8Δ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 μm. Characterization of this species from siz1Δ siz2Δ showed that it contains tandem copies of the 2 μm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 μm–encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 μm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 μm amplification.     

Margollus bokanicus n. sp. from Iran,the Fourth Species of a Rare Nematode Genus (Dorylaimida,Tylencholaimellidae)

Margollus bokanicus n. sp., collected from natural habitats in Khorasaneh district, Bokan, West Azarbaijan province, Iran, is described. Morphological and morphometric data are provided as well as drawings and light microscopy illustrations. The new species is characterized by a medium size body length (0.60 to 0.73 mm), labial and postlabial sclerotizations, lip region 7-μm wide, offset by constriction and long neck (167 to 207 μm), long pharyngeal basal bulb (27 to 36 μm) or 16% to 17% of total neck length, female genital system monodelphic–opisthodelphic, anterior branch reduced to a uterine sac (26–29 μm) or 1.1 to 1.3 times the body diameter, long posterior uterus (25–28 μm) or 1.1 to 1.3 times the body diameter, V = 40 to 47, cylindroid female tail (17 to 24 μm, c = 31 to 38, c’ = 1.1 to 1.4), and males unknown. This taxon is easily distinguishable from other Margollus species by its smaller general size and more posterior vulva. A compendium of Margollus species is also presented.     

Water-to-Air Transfer and Enrichment of Bacteria in Drops from Bursting Bubbles

An electrostatic induction technique was used to determine both drop size distribution and concentration of bacteria in the film drops produced by bubbles bursting at the surface of a suspension of Serratia marcescens. Film drops are produced from the collapse of the thin film of water that just before bursting separates the air in the bubble from the atmosphere. Bubbles of 1.7-mm diameter produced from 10 to 20 film drops which ranged from <2 μm to over 30 μm in diameter. Half the drops were <10 μm. For bubbles rising a distance of less than 2 cm through the bacterial suspension, bacterial enrichment factors in the drops were between 10 and 20. Electrostatic methods can be used to determine the enrichment of bacteria in film drops as a function of bubble size and distance of rise through the bacterial suspension.     

A new species of plasmodiidae (Coccidia: Hemosporidia) from the blood of the skink Scincus hemprichii (Scincidae: Reptilia) in Saudi Arabia

Fallisia arabica n. sp. was described from peripheral blood smears of the Skink lizard, Scincus hemprichii from Jazan Province in the southwest of Saudi Arabia. Schizogony and gametogony take place within neutrophils in the peripheral blood of the host. Mature schizont is rosette shaped 17.5 ± 4.1 × 17.0 ± 3.9 μm, with a L/W ratio of 1.03(1.02–1.05) μm and produces 24(18–26) merozoites. Young gametocytes are ellipsoidal, 5.5 ± 0.8 × 3.6 ± 0.5 μm, with a L/W of 1.53(1.44–1.61) μm. Mature macrogametocytes are ellipsoidal, 9.7 ± 1.2 × 7.8 ± 1.0 μm, with a L/W of 1.24(1.21–1.34) μm and microgametocytes are ellipsoidal, 7.0 ± 1.1 × 6.8 ± 0.9 μm. with a L/W of 1.03(1.01–1.10) μm. In comparison to the described Fallisia species, this new taxon has rosette schizonts and is larger than F. dominicensis, in Hispaniola, F. bipocrati, F. poecilopi, in Panama, F. thecadactyli in Venezuela, and F. effusa, F. simplex, F. modesta, in Brazil. F. arabica has fewer merozoites than F. effusa, F. poecilopi, F. thecadactyli and F. siamense in Thailand. This new species has more merozoites than F. dominicensis and F. modesta. All of these species belong to diverse saurian families (Agamidae, Gekkonidae, Polychrotidae, Scincidae and Teiidae) parasitize only thrombocytes or lymphocytes and some species parasitize immature erythroid cells and leucocytes.     

Obesity Is Associated with Lower Coronary Microvascular Density

Background

Obesity is associated with diastolic dysfunction, lower maximal myocardial blood flow, impaired myocardial metabolism and increased risk of heart failure. We examined the association between obesity, left ventricular filling pressure and myocardial structure.

Methods

We performed histological analysis of non-ischemic myocardium from 57 patients (46 men and 11 women) undergoing coronary artery bypass graft surgery who did not have previous cardiac surgery, myocardial infarction, heart failure, atrial fibrillation or loop diuretic therapy.

Results

Non-obese (body mass index, BMI, ≤30 kg/m², n=33) and obese patients (BMI >30 kg/m², n=24) did not differ with respect to myocardial total, interstitial or perivascular fibrosis, arteriolar dimensions, or cardiomyocyte width. Obese patients had lower capillary length density (1145±239, mean±SD, vs. 1371±333 mm/mm³, P=0.007) and higher diffusion radius (16.9±1.5 vs. 15.6±2.0 μm, P=0.012), in comparison with non-obese patients. However, the diffusion radius/cardiomyocyte width ratio of obese patients (0.73±0.11 μm/μm) was not significantly different from that of non-obese patients (0.71±0.11 μm/μm), suggesting that differences in cardiomyocyte width explained in part the differences in capillary length density and diffusion radius between non-obese and obese patients. Increased BMI was associated with increased pulmonary capillary wedge pressure (PCWP, P<0.0001), and lower capillary length density was associated with both increased BMI (P=0.043) and increased PCWP (P=0.016).

Conclusions

Obesity and its accompanying increase in left ventricular filling pressure were associated with lower coronary microvascular density, which may contribute to the lower maximal myocardial blood flow, impaired myocardial metabolism, diastolic dysfunction and higher risk of heart failure in obese individuals.     

Restricted Diffusion in Biophysical Systems: Experiment

The pulsed-gradient spin echo nuclear magnetic resonance (PGSENMR) technique was used to measure restricted diffusion of water in three types of animal tissue: human blood plasma and red cells; rat and rabbit heart; rat and rabbit liver. Characteristic lengths (L) for restriction of diffusion are estimated from dependence on the measuring time. Limitations on the range of observable restrictive lengths (1.5-15 μm) are discussed.

The decrease in diffusivity due to 1 μm alumina powder (volume fraction = 0.18) in glycerin/water mixtures agrees with the Wang theory assuming spherical particles and no hydration. The characteristic length (L 4 μm) is larger than the particle size (1 μm) or separation (1.8 μm). Comparison of the diffusivities in tissues at short diffusion times with the Wang theory indicates some bound or trapped water.

For packed red blood cells, a restriction (L 2.3 μm) was attributed tothe red cell membrane. A permeability p 0.014 cm/s may be estimated from the decrease in diffusivity. Average values of diffusivity ratio in heart were: 0.36 ± 0.02 for rat; and 0.26 ± 0.03 for rabbit; and in liver: 0.25 ± 0.01 for rat; 0.25 ± .04 for 10-day old rabbit; and 0.195 ± 0.03 for 2-yr old rabbit. A restriction (L 2.7 μm) in rat liver probably results from the mitochondria.

     

Clinical Factors Associated with Lamina Cribrosa Thickness in Patients with Glaucoma,as Measured with Swept Source Optical Coherence Tomography

PurposeTo investigate the influence of various risk factors on thinning of the lamina cribrosa (LC), as measured with swept-source optical coherence tomography (SS-OCT; Topcon).MethodsThis retrospective study comprised 150 eyes of 150 patients: 22 normal subjects, 28 preperimetric glaucoma (PPG) patients, and 100 open-angle glaucoma patients. Average LC thickness was determined in a 3 x 3 mm cube scan of the optic disc, over which a 4 x 4 grid of 16 points was superimposed (interpoint distance: 175 μm), centered on the circular Bruch’s membrane opening. The borders of the LC were defined as the visible limits of the LC pores. The correlation of LC thickness with Humphrey field analyzer-measured mean deviation (MD; SITA standard 24–2), circumpapillary retinal nerve fiber layer thickness (cpRNFLT), the vertical cup-to-disc (C/D) ratio, and tissue mean blur rate (MBR) was determined with Spearman''s rank correlation coefficient. The relationship of LC thickness with age, axial length, intraocular pressure (IOP), MD, the vertical C/D ratio, central corneal thickness (CCT), and tissue MBR was determined with multiple regression analysis. Average LC thickness and the correlation between LC thickness and MD were compared in patients with the glaucomatous enlargement (GE) optic disc type and those with non-GE disc types, as classified with Nicolela’s method.ResultsWe found that average LC thickness in the 16 grid points was significantly associated with overall LC thickness (r = 0.77, P < 0.001). The measurement time for this area was 12.4 ± 2.4 minutes. Average LC thickness in this area had a correlation coefficient of 0.57 with cpRNFLT (P < 0.001) and 0.46 (P < 0.001) with MD. Average LC thickness differed significantly between the groups (normal: 268 ± 23 μm, PPG: 248 ± 13 μm, OAG: 233 ± 20 μm). Multiple regression analysis showed that MD (β = 0.29, P = 0.013), vertical C/D ratio (β = -0.25, P = 0.020) and tissue MBR (β = 0.20, P = 0.034) were independent variables significantly affecting LC thickness, but age, axial length, IOP, and CCT were not. LC thickness was significantly lower in the GE patients (233.9 ± 17.3 μm) than the non-GE patients (243.6 ± 19.5 μm, P = 0.040). The correlation coefficient between MD and LC thickness was 0.58 (P < 0.001) in the GE patients and 0.39 (P = 0.013) in the non-GE patients.ConclusionCupping formation and tissue blood flow were independently correlated to LC thinning. Glaucoma patients with the GE disc type, who predominantly have large cupping, had lower LC thickness even with similar glaucoma severity.     

Diffuse Optical Characterization of the Healthy Human Thyroid Tissue and Two Pathological Case Studies

The in vivo optical and hemodynamic properties of the healthy (n = 22) and pathological (n = 2) human thyroid tissue were measured non-invasively using a custom time-resolved spectroscopy (TRS) and diffuse correlation spectroscopy (DCS) system. Medical ultrasound was used to guide the placement of the hand-held hybrid optical probe. TRS measured the absorption and reduced scattering coefficients (μ_a, μ_s′) at three wavelengths (690, 785 and 830 nm) to derive total hemoglobin concentration (THC) and oxygen saturation (StO₂). DCS measured the microvascular blood flow index (BFI). Their dependencies on physiological and clinical parameters and positions along the thyroid were investigated and compared to the surrounding sternocleidomastoid muscle. The THC in the thyroid ranged from 131.9 μM to 144.8 μM, showing a 25–44% increase compared to the surrounding sternocleidomastoid muscle tissue. The blood flow was significantly higher in the thyroid (BFI_thyroid = 16.0 × 10^-9 cm²/s) compared to the muscle (BFI_muscle = 7.8 × 10^-9 cm²/s), while StO₂ showed a small (StO_{2, muscle} = 63.8% to StO_{2, thyroid} = 68.4%), yet significant difference. Two case studies with thyroid nodules underwent the same measurement protocol prior to thyroidectomy. Their THC and BFI reached values around 226.5 μM and 62.8 × 10^-9 cm²/s respectively showing a clear contrast to the nodule-free thyroid tissue as well as the general population. The initial characterization of the healthy and pathologic human thyroid tissue lays the ground work for the future investigation on the use of diffuse optics in thyroid cancer screening.     

Altered Hematopoiesis in Mice Lacking DNA Polymerase μ Is Due to Inefficient Double-Strand Break Repair

Polymerase mu (Polμ) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ^−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.     

Penetration of Rhizopus oligosporus into Soybeans in Tempeh

Histological observations were made on the penetration of hyphae of Rhizopus oligosporus into soybean cotyledons in tempeh, an Indonesian soybean food. Hyphal penetrations averaged one per 1,400 μm² (±390 μm²) on the curved (outer) cotyledon surface and one per 1,010 μm² (±340 μm²) on the flat (inner) one. Hyphae infiltrated to a depth of 742 μm, or about 25% of the average width of a soybean cotyledon. This previously unreported degree of penetration offers partial explanation for the rapid physical and chemical changes in soybeans during tempeh fermentation.