Insulin-like factor 3 levels in amniotic fluid of human male fetuses

BACKGROUND: Rodent studies suggest that the peptide hormone insulin-like factor 3 (Insl3) made by the fetal testis is responsible for the first transabdominal phase of testicular descent, and may be affected by xenobiotics to disrupt male reproductive tract development. To date, there is very little information on the production of INSL3 by the human fetus during gestation. The objective of the present study was to determine the concentrations and time course during pregnancy of INSL3 and testosterone production in human fetuses and their associations with maternal characteristics, pregnancy complications and outcome. METHODS: This is a retrospective cohort study in which women who contributed amniotic fluid specimens to a bank from 2003-2006 were followed to determine their pregnancy complications and pregnancy outcome. Amniotic fluid specimens were collected from the Reproductive Genetics Laboratory of the Hospital of the University of Pennsylvania subsequent to routine amniocentesis. INSL3 and total testosterone levels were measured in amniotic fluid (from n = 50 female, n = 237 male fetuses) by validated immunoassays and correlated with maternal characteristics, pregnancy complications and outcomes. RESULTS: INSL3 was only detectable in amniotic fluid from male fetuses, and highest levels occurred from weeks 15-17 of gestation. INSL3 concentration was positively associated with increased birth weight, the occurrence of pre-eclampsia and advanced maternal age, but not with testosterone levels. CONCLUSIONS: INSL3 concentration in human amniotic fluid is potentially predictive of fetal sex and pre-eclampsia, and presumably reflects the functioning of the fetal Leydig cell population.     

An enhanced HOWFARLESS option for DOSXYZnrc simulations of slab geometries

The Monte Carlo code DOSXYZnrc is a valuable instrument for calculating absorbed dose within a three-dimensional Cartesian geometry. DOSXYZnrc includes several variance reduction techniques used to increase the efficiency of the Monte Carlo calculation. One such technique is HOWFARLESS which is used to increase the efficiency of beam commissioning calculations in homogeneous phantoms. The authors present an enhanced version of HOWFARLESS which extends the application to include phantoms inhomogeneous in one dimension. When the enhanced HOWFARLESS was used, efficiency increases as high as 14 times were observed without any loss in dose accuracy. The efficiency gains of an enhanced HOWFARLESS simulation was found to be dependent on both slab geometry and slab density. As the number of two-dimensional voxel layers per slab increases, so does the efficiency gain. Also, as the mass density of a slab is decreased, the efficiency gains increase.     

Relaxation times of skeletal muscle metabolites at 7T

Purpose

To demonstrate the feasibility of quantitatively evaluating and measuring T₁ and T₂ relaxation times of human tibialis anterior (TA) muscles metabolites in vivo at 7T and to compare these results with those of 3T.

Materials and Methods

A model lipid phantom (corn oil) and healthy volunteers (n = 4, mean ± SD age 35.6 ± 5.6 years) were scanned on 3T and 7T whole‐body MR scanners. A voxel of 10 × 10 × 10 mm³ was positioned on the lipid phantom and right calf TA muscles using the single‐voxel stimulated echo acquisition mode (STEAM) pulse sequence. All magnetic resonance spectroscopy (MRS) data were processed with Java‐based Magnetic Resonance User Interface (JMRUI) using Hankel Lanczos Singular Value Decomposition (HLSVD) filtering to remove the residual water signal.

Results

T₁ shows a steady increase while T₂ shows a slight decrease with B₀ and the spectra show larger spectral resolution at 7T than at 3T in the lipid phantom. T₁ values of all the metabolites are higher, while T₂ values are slightly lower at 7T than those of 3T compared to reported results in TA. The maximum percentage of increase in T₁ is about ≈488%, the maximum percentage of decrease in T₂ is about ≈65%.

Conclusion

The preliminary results can potentially be used for calculating relaxation correction factors required for absolute quantitation of skeletal muscle metabolite concentrations and for further protocol and sequence optimization. J. Magn. Reson. Imaging 2009;29:1457–1464. © 2009 Wiley‐Liss, Inc.     

Evaluation of tracheal intubation difficulty in patients with cervical spine immobilization: fiberoptic (WuScope) versus conventional laryngoscopy.

BACKGROUND: The WuScope is a rigid, fiberoptic laryngoscope designed to facilitate tracheal intubation without the need for head extension. The study evaluated the WuScope in anesthetized patients with neck immobilization. METHODS: Patients were randomized to one of two groups: those receiving fiberoptic laryngoscopy (WuScope, n = 43) and those receiving conventional laryngoscopy (Macintosh blade, n = 44). Manual in-line stablization of the cervical spine was done during intubation. Seven parameters of intubation difficulty were measured (providing an intubation difficulty scale score): number of operators, number of attempts, number of techniques, Cormack view, lifting force, laryngeal pressure, and vocal cord position. RESULTS: Successful intubation occurred in 95% of patients in the fiberoptic group and in 93% of patients in the conventional group. There were no differences in number of attempts. In the fiberoptic group, 79% of patients had an intubation difficulty scale score of 0, representing an ideal intubation: that is, one performed by the first operator on the first attempt using the first technique with full glottic visualization. Only 18% of patients in the conventional group had an intubation difficulty scale score of 0 (P < 0.001). More patients had Cormack grade 3 or 4 views with conventional than with fiberoptic laryngoscopy (39 vs. 2%, P < 0.001). Intubation times in patients with one attempt were slightly longer in the fiberoptic (median, 25th-75th percentiles: 30, 23-53 s) compared with the conventional group (24, 17-30 s, P < 0.05). Corresponding times in patients requiring > one attempt were 155 (range, 112-201) s and 141 (range, 95-186) s in the fiberoptic and conventional groups, respectively (P value not significant). CONCLUSIONS: Compared with conventional laryngoscopy, tracheal intubation using the fiberoptic laryngoscope was associated with lower intubation difficulty scale scores and better views of the laryngeal aperture in patients with cervical imnmobilization. However, there were no differences in success rates or number of intubation attempts.     

USP10 Promotes Fibronectin Recycling,Secretion, and Organization

PurposeIntegrins play a central role in myofibroblast pathological adhesion, over-contraction, and TGFβ activation. Previously, we demonstrated that after corneal wounding, αv integrins are protected from intracellular degradation by upregulation of the deubiquitinase USP10, leading to cell-surface integrin accumulation. Because integrins bind to and internalize extracellular matrix (ECM), we tested whether extracellular fibronectin (FN) accumulation can result from an increase in integrin and matrix recycling in primary human corneal fibroblasts (HCFs).MethodsPrimary HCFs were isolated from cadaver eyes. HCFs were transfected with either USP10 cDNA or control cDNA by nucleofection. Internalized FN was quantified with a FN ELISA. Recycled extracellular integrin and FN were detected with streptavidin-488 by live cell confocal microscopy (Zeiss LSM 780). Endogenous FN extra domain A was detected by immunocytochemistry. Cell size and removal of FN from the cell surface was determined by flow cytometry.ResultsUSP10 overexpression increased α5β1 (1.9-fold; P < 0.001) and αv (1.7-fold; P < 0.05) integrin recycling, with a concomitant increase in biotinylated FN internalization (2.1-fold; P < 0.05) and recycling over 4 days (1.7–2.2-fold; P < 0.05). The dependence of FN recycling on integrins was demonstrated by α5β1 and αv integrin blocking antibodies, which, compared with control IgG, decreased biotinylated FN recycling (62% and 84%, respectively; P < 0.05). Overall, we established that extracellular FN was composed of approximately 1/3 recycled biotinylated FN and 2/3 endogenously secreted FN.ConclusionsOur data suggest that reduced integrin degradation with a subsequent increase in integrin/FN recycling after wounding may be a newly identified mechanism for the characteristic accumulation of ECM in corneal scar tissue.keywords: myofibroblast, integrin, fibrosis, scarring, wound healing, cornea, deubiquitinase, fibronectin

Scarring in the eye can result in visual disability or blindness. In particular, because the cornea is transparent, a scar severely impacts vision.¹ For scientific discovery of scarring mechanisms, the cornea is a very useful model for studying wound healing, as scarring can be easily evaluated.^2,3 Corneal stromal wounding induces an influx of myofibroblasts and immune cells into the cornea.^4,5 The persistence of pathological myofibroblasts and fibrotic matrix forms a scar, blocking transmission of light.^6,7 Integrin-mediated adhesion promotes myofibroblast differentiation by increasing cell adhesion and cellular tension, required for the assembly of alpha-smooth muscle actin (α-SMA) stress fibers characteristic of myofibroblasts.^6,8 Integrins also activate latent, matrix-associated endogenous TGFβ by binding to the arginine–glycine–aspartic acid (RGD) domain in its latency-associated peptide.^9–11 FN-binding integrins (αv and α5β1) in particular are strongly associated with fibrotic outcomes.^9,12–18Our previous work discovered that the deubiquitinase (DUB) USP10 gene and protein expression were increased in human corneal myofibroblasts.¹³ DUBs remove ubiquitin from proteins, preventing degradation. We found that USP10 is a DUB for integrin subunits β1 and β5 but not on β3.¹³ (The αv integrin subunit is not directly ubiquitinated but is degraded with the β subunit).^19,20 Correspondingly, knockdown of USP10 gene expression increased ubiquitination of integrin β1 and β5 subunits, leading to decreased αv/β1/β5 protein levels, whereas gain of USP10 expression increased these protein levels without altering integrin gene expression.¹³ As a result of cell-surface integrin accumulation, TGFβ is activated, leading to increased gene expression and organization of the fibrotic markers, α-SMA and fibronectin extra domain A (FN-EDA).¹³ Blocking either TGFβ signaling or cell-surface αv integrins after USP10 overexpression prevented or reduced these fibrotic markers, respectively.¹³ Furthermore, knockdown of USP10 with USP10-targeting siRNA after wounding in an ex vivo pig cornea organ culture model and in an in vivo rabbit corneal model significantly reduced the induction of fibrotic markers and promoted regenerative healing^13,21,22 These data suggest that controlling integrin levels via DUB expression is a novel method to control scarring and fibrosis.Here, we have continued this work by testing if the USP10-mediated increase in integrins on the cell surface after wounding is an undiscovered mechanism for increasing extracellular matrix, a hallmark of scarring and fibrosis. We have focused this study on α5β1 and αv integrins and the matrix molecule, fibronectin. In a stepwise manner, FN secreted by cells is organized to assemble into fibrils (fibrillogenesis).²³ As assembly progresses, short detergent-soluble fibrils are converted into a dense detergent-insoluble fibrillar network.²⁴ The FN extracellular matrix is a dynamic scaffold that is a central player in cell repair, adhesion, migration, and invasion. The αv integrins, along with the classical FN receptor α5β1 integrin, bind to and coordinate the organization and endocytosis of FN through the binding of FN RGD domains.²⁵ Using single-cell force microscopy, it was demonstrated that αv integrins bind first to FN, signaling to α5β1 to form additional adhesion sites. This crosstalk strengthens adhesions to FN.²⁶ Integrins also mediate the endocytosis of FN,²⁷ but large organized FN fibrils cannot be endocytosed. Membrane-type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound MMP, plays a key role in the extracellular FN cleavage that is necessary for FN endocytosis and subsequent intracellular degradation^28,29 or recycling back to the cell surface.^30,31Disrupted FN homeostasis leading to a buildup of extracellular FN and, specifically, the fibrotic cellular splice variant, FN-EDA, is linked to the activation of TGFβ and a wide range of disease pathologies, including cardiac, liver, kidney, and dermal fibrosis, as well as glaucoma.^32–38 Here, we demonstrate that overexpression of the DUB USP10 increases α5β1 and αv integrin recycling to the cell surface. This increase in integrin recycling also promotes endocytosis and subsequent recycling of FN. Together, these data demonstrate that integrin ubiquitination status affects not only the recycling of integrin to the cell surface but also the recycling of integrin-bound matrix.     

Minimally Invasive Thyroid Surgery for Single Nodules: An Evidence-based Review of the Lateral Mini-incision Technique

Background

Minimally invasive thyroidectomy techniques are being developed in an effort to minimize pain, shorten the length of hospital stay, and improve cosmesis. Various minimally invasive thyroid surgery (MITS) techniques have been shown to be safe and feasible with some benefits in terms of cosmesis and pain outcomes; however, no single technique has been broadly accepted. This study was designed to review the evidence in relation to MITS and our experience with the direct lateral mini-incision technique.

Methods

A review of literature published until December 2007 on minimally invasive thyroidectomy techniques was undertaken. Three issues were addressed: 1) Does MITS provide any benefit compared with conventional open thyroidectomy? 2) Is there any advantage to the use of endoscopic or video-assisted techniques compared with the direct mini-incision technique? 3) Is the lateral mini-incision technique safe and efficacious? Additional data in relation to the above issues was derived from a retrospective cohort study of patients undergoing mini-incision thyroid surgery within our unit.

Results

Issue 1: Five prospective randomized studies and eight studies at a lower level of evidence have demonstrated consistent advantages of MITS compared with open thyroid surgery in terms of reduced pain and improved cosmesis with equivalent operative safety. Issue 2: In compiling four level III and IV studies that compared open and video-assisted minimally invasive surgery, there do not seem to be significant differences in patient satisfaction with the incision. The video-assisted approaches require significantly longer operative times but also seem to be less painful. Issue 3: Three cohort studies (level IV) have demonstrated that the lateral mini-incision technique is both safe and efficacious compared with open surgery for hemi-thyroidectomy. Data from our cohort study of 1281 patients (open hemi-thyroidectomy 1054 vs. MITS 227) confirmed MITS to be a safe and effective procedure. The rate of postoperative hematoma formation and wound infection was equivalent between groups. The rate of permanent recurrent laryngeal nerve injury was 0.4% for MITS and 0.3% for CHT and not significantly different (p = 0.7).

Conclusions

MITS has demonstrated advantages over conventional open approaches for both hemi- and total thyroidectomy and the benefits do not depend on the open or video-assisted approach. For thyroid lobectomies, the lateral mini-incision approach can be performed with an operative time and postoperative complication profile equivalent to conventional hemi-thyroidectomy while providing excellent cosmesis with a 2–3 cm scar.     

Routine level VI lymph node dissection for papillary thyroid cancer: surgical technique

Total thyroidectomy is the treatment of choice for clinically significant papillary thyroid cancer (PTC); however, 10-15% develop palpable local recurrence in the cervical lymph nodes. Metastases in the cervical lymph nodes account for 75% of loco-regional recurrence and up to 50% of these patients eventually die of their disease. It is generally accepted that surgical excision of grossly involved lymph node disease should be carried out. The role of routine lymph node dissection, however, is greeted with far more controversy. Regional lymph node metastases have been shown to be associated with more frequent tumour recurrence. Not only is recurrence associated with increased disease-related mortality, but recent data have shown that the presence of involved lymph nodes is associated with adverse survival. Additionally, there have been significant changes to the way patients are managed after treatment for PTC in recent years. Surveillance previously relied on clinical assessment and radioiodine scans whereas now the use of serum thyroglobulin and high-resolution ultrasound are the standard as evidenced by recommendations by the American Thyroid Association. These techniques have greater sensitivity and subsequently lymph node metastases are being detected earlier and more frequently. This has led to a paradigm shift in the aims of treatment of PTC, from a focus on survival data to a focus on disease-free status. Routine central neck lymph node dissection can be carried out with no increased morbidity and can achieve lower 6-month stimulated thyroglobulin levels when compared with total thyroidectomy alone. Routine ipsilateral level VI lymph node dissection in addition to total thyroidectomy should be carried out for the management of clinically significant PTC.     

NEW SURGICAL CONSULTANTS: IS THERE A LEARNING CURVE?

The purpose of this study was to determine whether newly established surgeons who have completed dedicated post-fellowship training are able to achieve surgical outcomes comparable to their more experienced peers. A cross-sectional study of consecutive patients undergoing total thyroidectomy (TT) or completion thyroidectomy was carried out. Outcomes measured included unplanned return to the operating theatre, postoperative infection, permanent recurrent laryngeal nerve (RLN) injury and permanent hypoparathyroidism. Outcomes were categorized according to whether surgery was carried out by an established surgeon (ES) or a newly appointed surgeon (NAS). Eight hundred and nine TT and completion thyroidectomy procedures were carried out in the period January 2002 to December 2004. Of these, 515 (64%) were carried out by ES and 294 (36%) were carried out by NAS. The overall rate of permanent hypoparathyroidism and RLN injury was 1.4% (12/809) and 0.6% (5/809), respectively. The rate of permanent hypoparathyroidism was not significantly different between the two categories of surgeon (ES 1.35% vs NAS 1.7%; P = 0.7). The incidence of permanent RLN injury was not different between the two groups (ES 0.8% vs NAS 0.3%; P = 0.4). For NAS, the rate of permanent RLN injury for the first two years of independent practice did not differ significantly from 3 to 4 years of practice (0/123 vs 1/171; P = 0.4). Indications for surgery between the two groups were similar, with ES carrying out TT for benign goitre in 42% and cancer in 28%, and NAS 44 and 32%, respectively. Surgical outcomes for the newly established endocrine surgeon following subspecialty training are equivalent to those achieved by more experienced surgeons.