Does implant removal of superior clavicle plate osteosynthesis affect the functional outcome: a prospective trial.

BACKGROUND: Elective implant removal (IR) accounts for up to 30% of all orthopaedic surgeries. While there is general acceptance about the need of implant removal for obvious reasons, such as infections or implant failure, little is known about the beneficial aspects in cases of minor reasons such as patients' wish for IR. Therefore, we initiated this study to define patients' benefit of elective implant removal following plate osteosynthesis of displaced clavicle fractures. PATIENTS AND METHODS: Prospective evaluation of patients was conducted before implant removal and 6 weeks postoperative. Subjective and objective criteria included pain rating on a visual analogue scale (VAS) and active range of motion (ROM) pre- and 6 weeks postoperative. Functional scoring included Constant-Murley Score, DASH (Disabilities of Arm, Shoulder and Hand Score), MSQ (Munich Shoulder Questionnaire) and SPADI (Shoulder Pain and Disability Index). RESULTS: 37 patients were prospectively enrolled in this study and implant removal was performed after 16 ± 6.1 months. No re-fractures nor other complications were detected during routine follow up. Functional outcome increased through all scores (Constant score 73.3 ± 14.6 preoperative to 87.4 ± 12.0 postoperative (p = 0.000), MSQ 85.0 ± 7.3 preoperative to 91.8 ± 9.0 postoperative (p = 0.005), DASH Score 7.4 ± 8.2 preoperative to 5.7 ± 9.5 postoperative (p = 0.414), SPADI 93.4 ± 6.6 preoperative to 94.0 ± 10.1 postoperative (p = 0.734). CONCLUSIONS: Discomfort during daily activities or performing sports as well as limited range of motion were the main reasons for patients' wish for implant removal. We found increased functional outcome parameters and decreased irritation after implant removal. Therefore we suggest implant removal in case of patients' wish and completed fracture consolidation. TRIAL REGISTRATION: Trial registration no: NCT04343118, Retrospective registered: www.clinicaltrials.gov .

[Functional outcome of 111 metatarsal fractures following conservative in comparison to operative treatment]. / Funktionelles Outcome nach konservativer im Vergleich zu operativer Therapie von 111 Mittelfußfrakturen.

BACKGROUND: Fractures of the metatarsal bones are common injuries of the foot and particularly occur in patients aged 40-50 years. Especially multiple metatarsal fractures can lead to permanent limitations. Therefore, the aim of this study was to investigate the functional outcome of metatarsal fractures after conservative and surgical treatment using a validated self-reported patient-based outcome questionnaire. MATERIAL AND METHODS: All patients suffering from metatarsal fractures between 2003 and 2015 were enrolled in this retrospective analysis. The following data were collected: demographic data, AO classification, treatment, reoperation rate and the foot and ankle outcome score (FAOS). For outcome analysis, the nonparametric Mann-Whitney U­test and Fisher's exact test were performed. RESULTS: In total the functional outcome of 111 patients with metatarsal fractures were analyzed, 81 patients suffered of an isolated metatarsal fracture and 30 of multiple fractures. The mean age of the patients was 45 ± 15.2 years with a total of 48 men (43%) and 63 women (57%). Patients with an isolated metatarsal fracture had an FAOS of 88 ± 17.1, while patients with multiple metatarsal fractures achieved an FAOS of 78 ± 17.7 (p = 0.046). In the group of isolated metatarsal fractures 43 patients (53%) were surgically treated and of these 36 patients showed a type C fracture (84%). In the group of multiple metatarsal fractures 16 patients (53%) underwent operative treatment. CONCLUSION: Overall, the functional outcome of isolated metatarsal fractures following operative as well as conservative treatment is good to very good. Simple fractures can be successfully treated conservatively and complex multifragment fractures can be safely managed surgically. If more than one metatarsal bone is fractured, the functional outcome is significantly worse with patients reporting lasting limitations involving the range of motion and stiffness.

[Mobile C-arm-Radiation exposure and workflow killer? : Potential of an innovative assistance system for intraoperative positioning]. / Mobiler C-Bogen ­ Strahlenbelastung und Workflow-Killer? : Potenzial eines innovativen Assistenzsystems für die intraoperative Positionierung.

Despite its versatile applicability the intraoperative use of a mobile C­arm is often problematic and potentially associated with increased radiation exposure for both the patient and the personnel. In particular, the correct positioning for adequate imaging can become a problem as the nonsterile circulating nurse has to coordinate the various maneuvers together with the surgeon without having a good view of the surgical field. The sluggishness of the equipment and the intraoperative setting (sterile borders, additional hardware, etc.) pose further challenges. A light detection and ranging (LIDAR)-based assistance system shows promise to provide accurate and intuitive repositioning support as part of an initial series of experimental trials. For this purpose, the sensors are attached to the C­arm base unit and enable navigation of the device in the operating room to a stored target position using a simultaneous localization and mapping (SLAM) algorithm. An improvement of the workflow as well as a reduction of radiation exposure represent the possible potential of this system. The advantages over other experimental approaches are the lack of external hardware and the ease of use without isolating the operator from the rest of the operating room environment; however, the suitability for daily use in the presence of additional interfering factors should be verified in further studies.

RAY-POS: a LIDAR-based assistance system for intraoperative repositioning of mobile C-arms without external aids.

PURPOSE: In current clinical practice, intraoperative repositioning of mobile C-arms is challenging due to a lack of visual cues and efficient guiding tools. This can be detrimental to the surgical workflow and lead to additional radiation burdens for both patient and personnel. To overcome this problem, we present our novel approach Lidar-based X-ray Positioning for Mobile C-arms (RAY-POS) for assisting circulating nurses during intraoperative C-arm repositioning without requiring external aids. METHODS: RAY-POS consists of a localization module and a graphical user interface for guiding the user back to a previously recorded C-Arm position. We conducted a systematic comparison of simultaneous localization and mapping (SLAM) algorithms using different attachment positions of light detection and ranging (LIDAR) sensors to benchmark localization performance within the operating room (OR). For two promising combinations, we conducted further end-to-end repositioning tests within a realistic OR setup. RESULTS: SLAM algorithm gmapping with a LIDAR sensor mounted 40 cm above the C-arm's horizontal unit performed best regarding localization accuracy and long-term stability. The distribution of the repositioning error yielded an effective standard deviation of 7.61 mm. CONCLUSION: We conclude that a proof-of-concept for LIDAR-based C-arm repositioning without external aids has been achieved. In future work, we mainly aim at extending the capabilities of our system and evaluating the usability together with clinicians.

Five amino acids in the innermost cavity of the substrate binding cleft of organic cation transporter 1 interact with extracellular and intracellular corticosterone.

We have shown previously that Leu447 and Gln448 in the transmembrane helix (TMH) 10 of rat organic cation transporter rOCT1 are critical for inhibition of cation uptake by corticosterone. Here, we tested whether the affinity of corticosterone is different when applied from the extracellular or intracellular side. The affinity of corticosterone was determined by measuring the inhibition of currents induced by tetraethylammonium(+) (TEA(+)) in Xenopus laevis oocytes expressing rOCT1. Either corticosterone and TEA(+) were added to the bath simultaneously or the oocytes were preincubated with corticosterone, washed, and TEA(+)-induced currents were determined subsequently. In mutant L447Y, K(i) values for extracellular and intracellular corticosterone were decreased, whereas in mutant Q448E, only the K(i) for intracellular corticosterone was changed. Modeling of the interaction of corticosterone with rOCT1 in the inward- or outward-facing conformation predicted direct binding to Leu447, Phe160 (TMH2), Trp218 (TMH4), Arg440 (TMH10), and Asp475 (TM11) from both sides. In mutant F160A, affinities for extracellular and intracellular corticosterone were increased, whereas maximal inhibition was reduced in W218F and R440K. In stably transfected epithelial cells, the affinities for inhibition of 1-methyl-4-phenyl-pyridinium(+) (MPP(+)) uptake by extracellular and intracellular corticosterone were decreased when Asp475 was replaced by glutamate. In mutants F160A, W218Y, R440K, and L447F, the affinities for MPP(+) uptake were changed, and in mutant D475E, the affinity for TEA(+) uptake was changed. The data suggest that Phe160, Trp218, Arg440, Leu447, and Asp475 are located within an innermost cavity of the binding cleft that is alternatingly exposed to the extracellular or intracellular side during substrate transport.

Transport of lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] and high-affinity interaction of nucleoside reverse transcriptase inhibitors with human organic cation transporters 1, 2, and 3.

Nucleoside reverse transcriptase inhibitors (NRTIs) need to enter cells to act against the HIV-1. Human organic cation transporters (hOCT1-3) are expressed and active in CD4+ T cells, the main target of HIV-1, and have been associated with antiviral uptake in different tissues. In this study, we examined whether NRTIs interact and are substrates of hOCT in cells stably expressing these transporters. Using [(3)H]N-methyl-4-phenylpyridinium, we found a high-affinity interaction among abacavir [[(1S,4R)-4-[2-amino-6-(cyclopropylamino)purin-9-yl]-cyclopent-2-enyl]methanol sulfate] (ABC); <0.08 nM], azidothymidine [3'-azido-3'-deoxythymidine (AZT); <0.4 nM], tenofovir disoproxil fumarate (<1.0 nM), and emtricitabine (<2.5 nM) and hOCTs. Using a wide range of concentrations of lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacyitidine (3TC)], we determined two different binding sites for hOCTs: a high-affinity site (K(d1) = 12.3-15.4 pM) and a low-affinity site (K(d2) = 1.9-3.4 mM). Measuring direct uptake of [(3)H]3TC and inhibition with hOCT substrates, we identified 3TC as a novel substrate for hOCT1, 2, and 3, with hOCT1 as the most efficient transporter (K(m) = 1.25 +/- 0.1 mM; V(max) = 10.40 +/- 0.32 nmol/mg protein/min; V(max)/K(m) = 8.32 +/- 0.40 microl/mg protein/min). In drug-drug interaction experiments, we analyzed cis-inhibition of [(3)H]3TC uptake by ABC and AZT and found that 40 to 50% was inhibited at low concentrations of the drugs (K(i) = 22-500 pM). These data reveal that NRTIs experience a high-affinity interaction with hOCTs, suggesting a putative role for these drugs as modulators of hOCT activity. Finally, 3TC is a novel substrate for hOCTs and the inhibition of its uptake at low concentrations of ABC and AZT could have implications for the pharmacokinetics of 3TC.

Long-term Results After Arthroscopic Repair of Isolated Subscapularis Tears.

BACKGROUND: Although some reports have presented short- to midterm results after arthroscopic repair of isolated subscapularis (SSC) tendon tears, long-term evaluation is still lacking. HYPOTHESIS: Long-term results after arthroscopic repair of isolated SSC tears are comparable with the functional and radiological short- to midterm outcomes described in the literature. STUDY DESIGN: Case series, Level of evidence, 4. METHODS: This study assessed 17 patients (5 females and 12 males; mean age, 45.6 years) with isolated SSC tears (Fox and Romeo classification types 2-4) who underwent all-arthroscopic suture anchor repair. The mean interval from symptom onset to the time of surgery was 5.3 months in 16 patients (94.1%). One patient (5.9%) was symptomatic for a prolonged period (104 months) before surgery. All patients were assessed with a clinical examination preoperatively. SSC function was investigated using specific clinical tests and common scoring systems, including Constant, American Shoulder and Elbow Surgeons (ASES), Disabilities of the Arm, Shoulder and Hand (DASH), and Simple Shoulder Test (SST) scores. At follow-up, muscular strength was evaluated using an electronic force-measuring plate. Structural integrity of the repair was assessed using magnetic resonance imaging (MRI). RESULTS: At a mean follow-up of 98.4 ± 19.9 months, the mean Constant score improved from 47.8 preoperatively to 74.2 postoperatively ( P = .001). Higher Constant ( P = .010) and ASES ( P = .001) scores were significantly associated with a shorter time from symptom onset to surgery. The size of the SSC lesion did not correlate with any clinical score outcome ( P = .476, .449, .985, and .823 for Constant, ASES, DASH, and SST scores, respectively). Three patients (17.6%) had persistent positive clinical test results (belly-press/lift-off). Compared with the uninjured contralateral side, SSC strength was significantly reduced in the belly-press position ( P = .031), although active internal ( P = .085) and external ( P = .093) rotation was not affected. In 1 patient, a rerupture was detected by MRI. Six patients had cranial SSC atrophy. Overall, 88.2% of patients were "very satisfied" or "satisfied" with their results. CONCLUSION: Arthroscopic repair of isolated SSC tears results in significant clinical improvements and enduring tendon integrity, although SSC strength remains reduced in the long term. Early surgical treatment seems to be a relevant factor allowing good shoulder function.

Cellular uptake of imatinib into leukemic cells is independent of human organic cation transporter 1 (OCT1).

PURPOSE: In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake. EXPERIMENTAL DESIGN: Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines. RESULTS: Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations. CONCLUSIONS: These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance.

Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications.

The body is equipped with broad-specificity transporters for the excretion and distribution of endogeneous organic cations and for the uptake, elimination and distribution of cationic drugs, toxins and environmental waste products. This group of transporters consists of the electrogenic cation transporters OCT1-3 (SLC22A1-3), the cation and carnitine transporters OCTN1 (SLC22A4), OCTN2 (SLC22A5) and OCT6 (SLC22A16), and the proton/cation antiporters MATE1, MATE2-K and MATE2-B. The transporters show broadly overlapping sites of expression in many tissues such as small intestine, liver, kidney, heart, skeletal muscle, placenta, lung, brain, cells of the immune system, and tumors. In epithelial cells they may be located in the basolateral or luminal membranes. Transcellular cation movement in small intestine, kidney and liver is mediated by the combined action of electrogenic OCT-type uptake systems and MATE-type efflux transporters that operate as cation/proton antiporters. Recent data showed that OCT-type transporters participate in the regulation of extracellular concentrations of neurotransmitters in brain, mediate the release of acetylcholine in non-neuronal cholinergic reactions, and are critically involved in the regulation of histamine release from basophils. The recent identification of polymorphisms in human OCTs and OCTNs allows the identification of patients with an increased risk for adverse drug reactions. Transport studies with expressed OCTs will help to optimize pharmacokinetics during development of new drugs.

Identification of cysteines in rat organic cation transporters rOCT1 (C322, C451) and rOCT2 (C451) critical for transport activity and substrate affinity.

Effects of the sulfhydryl reagent methylmethanethiosulfonate (MMTS) on functions of organic cation transporters (OCTs) were investigated. Currents induced by 10 mM choline [I(max(choline))] in Xenopus laevis oocytes expressing rat OCT1 (rOCT1) were increased four- to ninefold after 30-s incubation with 5 mM MMTS whereas I(max(choline)) by rat OCT2 was 70% decreased. MMTS activated the rOCT1 transporter within the plasma membrane without changing stoichiometry between translocated charge and cation. After modification of oocytes expressing rOCT1 or rOCT2 with MMTS, I(0.5(choline)) values for choline-induced currents were increased. For rOCT1 it was shown that MMTS increased I(0.5) values for different cations by different degrees. Mutagenesis of individual cysteine residues in rOCT1 revealed that modification of cysteine 322 in the large intracellular loop, and of cysteine 451 at the transition of the transmembrane alpha-helix (TMH) 10 to the short intracellular loop between the TMH 10 and 11 is responsible for the observed effects of MMTS. After replacement of cysteine 451 by methionine, the IC(50(choline)) for choline to inhibit MPP uptake by rOCT1 was increased whereas the I(0.5(choline)) value for choline-induced current remained unchanged. At variance, in double mutant Cys322Ser, Cys451Met, I(0.5(choline)) was increased compared with rOCT1 wild-type whereas in the single mutant Cys322Ser I(0.5(choline)) was not changed. The data suggest that modification of rOCT1 at cysteines 322 and 451 leads to an increase in turnover. They indicate that cysteine 451 in rOCT1 interacts with the large intracellular loop and that cysteine 451 in both rOCT1 and rOCT2 is critical for the affinity of choline.

Subtype-specific affinity for corticosterone of rat organic cation transporters rOCT1 and rOCT2 depends on three amino acids within the substrate binding region.

The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are approximately 150 and approximately 4 microM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane alpha helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the K(m) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 microM) uptake by corticosterone (24 +/- 4 microM) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 microM) uptake (5.3 +/- 1.7 microM). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.

Polyspecific cation transporters mediate luminal release of acetylcholine from bronchial epithelium.

In airway epithelia, non-neuronal cholinergic regulations have been described; however, the route for acetylcholine (ACh) release has not been verified. To investigate whether organic cation transporters (OCTs) serve this function, we studied the expression of OCTs in airway epithelia and their capability to translocate ACh. Using immunohistochemistry in rats and humans, OCT1, OCT2, and OCT3 were localized to the luminal membrane of ciliated epithelial cells. In humans, OCT2 showed the strongest expression in the luminal membrane. We expressed the OCT isoforms in oocytes of Xenopus laevis and measured uptake and efflux of ACh. Tracer flux measurements showed that ACh is transported by OCT1 and OCT2 but not by OCT3. Two-electrode-voltage-clamp measurements revealed that OCT2 mediates electrogenic uptake and efflux of ACh. For ACh uptake by human OCT2, a K(M) value of approximately 0.15 mM was determined. At -50 mV, ACh efflux by human OCT2 was trans-inhibited by micromolar concentrations of the inhalational glucocorticoid budesonide, which is used in treatment of asthma (K(i) approximately 2.7 microM). The data show that OCT1 and OCT2 mediate luminal ACh release in human airways and suggest that ACh release is blocked after inhalation of budesonide.

Different affinities of inhibitors to the outwardly and inwardly directed substrate binding site of organic cation transporter 2.

The rat organic cation transporter 2 (rOCT2) was expressed in Xenopus laevis oocytes and cation-induced outward and inward currents were measured in whole cells and giant patches using voltage clamp techniques. Tetrabutylammonium (TBuA) and corticosterone were identified as nontransported inhibitors that bind to the substrate binding site of rOCT2. They inhibited cation-induced currents from both membrane sides. Increased substrate concentrations could partially overcome the inhibition. At 0 mV, the affinity of TBuA from the extracellular side compared with the intracellular side of the membrane was 4-fold higher, whereas the affinity of corticosterone was 20-fold lower. The data suggest that the substrate binding site of rOCT2 is like a pocket containing overlapping binding domains for ligands. These binding domains may undergo separate structural changes. From the extracellular surface, the affinity for uncharged corticosterone was increased by making membrane potential more negative. This implies potential-dependent structural changes in the extracellular binding pocket and existence of a voltage sensor. Interestingly, at 0 mV, an 18-fold higher affinity was determined for trans-inhibition of choline efflux by corticosterone compared with cis-inhibition of choline uptake. This suggests an additional high affinity-conformation of the empty outwardly oriented substrate binding pocket. A model is proposed that describes how substrates and inhibitors might interact with rOCT2. The data provide a theoretical basis to understand drug-drug interactions at polyspecific transporters for organic cations.

The cation transporters rOCT1 and rOCT2 interact with bicarbonate but play only a minor role for amantadine uptake into rat renal proximal tubules.

In renal proximal tubules, the organic cation transporters rOCT1 and rOCT2 are supposed to mediate the first step in organic cation secretion. We investigated whether previously described differences in amantadine and tetraethylammonium (TEA) uptake into isolated renal proximal tubules could be explained by differences in their transport by rOCT1 and rOCT2. By expressing rOCT1 and rOCT2 in Xenopus oocytes and HEK 293 cells, we demonstrated that both transporters translocated amantadine. In Xenopus oocytes, the inhibitory potency of several rOCT1/2 inhibitors was similar for amantadine compared to TEA uptake and supports amantadine transport by rOCT1 and rOCT2. In proximal tubules, procainamide, quinine, cyanine(863), choline, and guanidine in concentrations that inhibit rOCT1/2-mediated TEA or amantadine uptake in Xenopus oocytes exhibited no effect on amantadine uptake. At variance, these inhibitors blocked TEA uptake into proximal tubules. Amantadine and TEA transport were sensitive to modulation by 25 mM bicarbonate. The effect of bicarbonate on organic cation transport was dependent on substrate (amantadine or TEA), cell system (oocytes, HEK 293 cells, or proximal tubules), and transporter (rOCT1 or rOCT2). In proximal tubules, only amantadine uptake was stimulated by bicarbonate. The data suggested that rat renal proximal tubules contain an organic cation transporter in addition to rOCT1 and rOCT2 that mediates amantadine uptake and requires bicarbonate for optimal function. TEA uptake by the basolateral membrane may be mediated mainly by rOCT1 and rOCT2, but these transporters may be in a different functional or regulatory state when expressed in cells or oocytes compared with expression in vivo.

A glucose sensor hiding in a family of transporters.

We have examined the expression and function of a previously undescribed human member (SGLT3/SLC5A4) of the sodium/glucose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na+-dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na+/glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.